EP2649452B1 - Method and kit for the in vitro diagnosis of prostate cancer - Google Patents

Method and kit for the in vitro diagnosis of prostate cancer Download PDF

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EP2649452B1
EP2649452B1 EP11811042.8A EP11811042A EP2649452B1 EP 2649452 B1 EP2649452 B1 EP 2649452B1 EP 11811042 A EP11811042 A EP 11811042A EP 2649452 B1 EP2649452 B1 EP 2649452B1
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directed against
antibodies
antibody
residue
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Yasemin ATAMAN-ÖNAL
Sandrine Busseret
Geneviève CHOQUET-KASTYLEVSKY
Emanuelle Gallet-Gorius
Gaspard Gervasi
Michèle GUILLOTTE
Céline HAMELIN
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Biomerieux SA
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069Reproductive system, e.g. ovaria, uterus, testes, prostate
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57434Specifically defined cancers of prostate
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/90Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701Details
    • G01N2333/4718Lipocortins

Definitions

  • the present invention relates to a method and a kit for the in vitro diagnosis of prostate cancer.
  • Prostate cancer is the most common type of cancer in men. Prostate cancer has the characteristic of evolving very slowly. Nevertheless, despite its moderate biological aggressiveness, compared to other types of cancer, it is the second most fatal cancer in men, after lung cancer, tied with colorectal cancer (1). The severity of the cancer depends on the extent of the tumor (local, with nearby or distant metastases) and the type of cancer cells, that is, the degree of malignancy. There are curative solutions, especially for early stages of cancer, and limited locally such as radiotherapy and / or surgery. It is therefore crucial to have a test that allows the earliest possible and reliable diagnosis of prostate cancer.
  • Prostate cancer can be tracked by the finding of increased concentration of a protein in the blood, prostate specific antigen or PSA.
  • PSA is produced by the prostate and is therefore a specific marker of the organ but it is not specific to the tumor. This is why a high result in a PSA quantification test does not necessarily mean that there is cancer.
  • an amount of more than four nanograms per milliliter of this protein in the blood is associated with prostate cancer in 25% of cases and another prostate disorder in 75% of cases, especially when benign hypertrophy (BPH), inflammation or infection of the prostate.
  • BPH benign hypertrophy
  • this test does not detect all cases of prostate cancer.
  • Annexin 3 (ANXA3) which belongs to the Annexines family.
  • the proteins of the Annexin family have the common characteristic of being able to bind to phospholipid membranes in the presence of calcium. To do this, the members of this protein family share structural similarities.
  • each Annexin contains in its protein sequence domains called "annexin repeat", which are 4 or 8 (4 for Annexin A3), and have a length of about 70 amino acids (2).
  • Each repetitive domain also referred to as the endonexin domain, is folded into 5 alpha helices and contains a characteristic motif of type 2 (GxGT- [38 residues] -D / E) which makes it possible to fix Ca2 + ions.
  • Annexins of the animal kingdom have variable N-terminal domains, which often carry the functional specificities of proteins. The crystallographic analyzes show that the secondary and tertiary structures of annexins are conserved even if the identity of the amino acid sequences does not exceed 45 to 55%.
  • the 4 repeat domains form a disk-like structure with a slightly convex surface on which the Ca 2+ binding sites are located and a concave surface where the N- and C-terminal ends of the protein are found. proximity.
  • Annexin A3 is a rare annexin, which is expressed only slightly or not at all by the majority of cell types. Conversely, it is very abundant in human neutrophils, where it accounts for about 1% of cytosolic proteins (3, 4). Initially, two isoforms of Annexin A3 have been described, one of 33 kDa of apparent molecular weight, predominantly present in neutrophils, the other of 36 kDa apparent molecular weight, found in monocytes. The difference in molecular weight between the two isoforms does not result from a post-translational modification such as phosphorylation or glycosylation. Very recently, in 2010, it was shown that an alternative splicing phenomenon could be a mechanism for explaining the presence of these two isoforms.
  • the authors identified two DNA variants complementary to Annexin A3: the first of 973 base pairs (bp) length encodes an entire protein of 323 amino acids corresponding to the 36 kDa isoform; the second with a length of 885 bp does not contain exon III following alternative splicing.
  • the open reading frame is interrupted, and the translation becomes possible only from the ATG codon present in exon IV.
  • the first 39 amino acids of the protein are not expressed, resulting in an N-terminal truncated protein of 284 amino acids, corresponding to the 33 kDa isoform (5).
  • Annexin A3 is recognized as a marker for prostate cancer, the methods for detecting it described so far are lacking in specificity because the antibodies used cross-react with other annexins ( WO 2006/125580 ), or lack sensitivity to detect it in a complex environment such as urine ( WO 2007/141043 ). In general, the prior methods lack robustness.
  • sequence of the first repeated domain of native human Annexin A3 is identified in SEQ ID NO: 1, the sequence of the fourth repeated domain of Annexin A3.
  • native human is identified in SEQ ID NO: 2
  • sequence of the second repeating domain of native human Annexin A3 is identified in SEQ ID NO: 3
  • sequence of the third repeating domain of native human Annexin A3 is identified in SEQ ID NO: 4.
  • sequence identified in SEQ ID NO: 5 corresponds to the amino acid sequence of complete native human Annexin A3 (UniProtKB Accession No. P12429).
  • the subject of the present invention is therefore a method for the in vitro diagnosis of prostate cancer according to which a urine sample to be analyzed is brought into contact with two antibodies, a capture antibody and a detection antibody, a of the two antibodies is directed against the first repeated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 1 and the other of the two antibodies is directed against the fourth replicated domain of native human Annexin A3 whose the sequence is identified in SEQ ID NO: 2.
  • antibodies which are directed against the fourth repeating domain of native human Annexin A3 there may be mentioned antibodies which are directed against an epitope whose amino acid sequence whose amino acid sequence comprises at least 7 consecutive amino acids and not more than 50, preferably no more than 45 consecutive amino acids of SEQ ID NO: 2.
  • the positions 6, 49, 7, 8 and 9 determined with respect to SEQ ID NO: 2 correspond respectively to the positions 263, 306, 264, 265 and 266 of the human Annexin A3 identified in SEQ ID NO: 5 which corresponds to the amino acid sequence of complete native Human Annexin A3 (UniProtKB Accession No. P12429).
  • the antibody directed against the first repeated domain of native human Annexin A3, whose sequence is identified in SEQ ID NO: 1, is the capture antibody and the antibody directed against the fourth repetitive domain of the Native human Annexin A3, whose sequence is identified in SEQ ID NO: 2, is the detection antibody.
  • the antibodies specific for the first repeated domain of human native Annexin A3 and specific for the fourth repetitive domain of human native Annexin A3, that is to say the capture and detection antibodies described above, are antibodies which exhibit a high affinity with an affinity constant of at least 10 -9 , preferably at least 10 -10 and which furthermore have a low dissociation constant of less than 2 10 -3 s -1 , more preferably lower at 5 10 -4 s -1 .
  • the antibodies are monoclonal antibodies and the preferred antibodies are the following antibodies: TGC42, TGC43 and TGC44 used as capture antibodies 13A12G4H2 and 1F10A6 used as detection antibodies.
  • TGC42, TGC43 and TGC44 used as capture antibodies 13A12G4H2 and 1F10A6 used as detection antibodies.
  • the preferred pair consisting of the TGC44 capture antibody and the detection antibody 13A12G4H2.
  • the invention also relates to an immunoassay kit for the in vitro diagnosis of prostate cancer in an urine sample to be analyzed comprising two antibodies, a capture antibody and a detection antibody, one of the two antibodies. being directed against the first repeated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 1 and the other of the two antibody antibodies being directed against the fourth replicated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 2 and an explanatory note.
  • the capture antibody is directed against the first repeated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 1 and the detection antibody directed against the fourth repeated domain of Annexin A3.
  • native human whose sequence is identified in SEQ ID NO: 2.
  • the capture and detection antibodies of the kit have the same characteristics as those described above for the process.
  • antibody is meant a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody or a fragment of said antibodies, in particular Fab, Fab 'fragments, F (ab ') 2, ScFv, Fv, Fd.
  • said antibodies must be specific for Annexin A3, that is to say that they do not cross-react with other annexins and that they are specific for the first repeated domain of Annexin A3 or specific to the fourth repeated domain of Annexin A3; the antibodies with the highest affinities and the weakest dissociation constants being the preferred antibodies.
  • the polyclonal antibodies can be obtained by immunizing an animal with the appropriate immunogen, followed by the recovery of the desired antibodies in purified form, by taking the serum of said animal, and separating said antibodies from the other constituents of the serum, in particular by chromatography. affinity on a column to which is attached an antigen specifically recognized by the antibodies.
  • the monoclonal antibodies can be obtained by the hybridoma technique whose general principle is recalled below.
  • an animal is immunized with the appropriate immunogen whose B cells are then capable of producing antibodies against this antigen.
  • These antibody-producing lymphocytes are then fused with "immortal" myeloma cells (murine in the example) to give rise to hybridomas.
  • the cells capable of producing a particular antibody and of multiplying indefinitely are then selected.
  • Each hybridoma is multiplied in the form of a clone, each leading to the production of a monoclonal antibody whose protein recognition properties can be tested, for example by ELISA, by immunoblot (Western blot) in one or two-dimensional, in immunofluorescence, or with a biosensor.
  • the monoclonal antibodies thus selected are subsequently purified, in particular according to the affinity chromatography technique described above.
  • the monoclonal antibodies can also be recombinant antibodies obtained by genetic engineering, by techniques well known to those skilled in the art.
  • the capture antibody is preferably fixed, directly or indirectly, on a solid support, for example a cone, a well a microtitration plate etc ...
  • Indirect detection systems can also be used, such as, for example, ligands capable of reacting with an anti-ligand.
  • the ligand / anti-ligand couples are well known to those skilled in the art, which is the case, for example, of the following pairs: biotin / streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, sugar / lectin. In this case, it is the ligand that carries the binding partner.
  • the anti-ligand can be detectable directly by the labeling reagents described in the preceding paragraph or be itself detectable by a ligand / anti-ligand.
  • the method of the invention is a "sandwich” type immunoassay carried out on a urine sample, in particular an "expressed” urine sample, that is to say a urine sample obtained after digital rectal examination.
  • mice Three days after the last injection, one of the immunized mice was sacrificed; blood and spleen were removed. Splenocytes obtained from the spleen were cultured with Sp2 / 0-Ag14 myeloma cells to fuse and immortalize, according to the protocol described by (8, 9). After a 12-14 day incubation period, the resulting hybridoma supernatants were screened for the presence of anti-ANXA3 antibodies using the ELISA test described in the previous paragraph. The immunogen (native ANXA3), recombinant Annexin A3 produced in E. coli, and various human cells expressing ANXA3 were used successively to screen the hybridoma supernatants. Positive hybridoma colonies were subcloned twice according to the limiting dilution technique, well known to those skilled in the art.
  • the anti-annexin monoclonal antibodies A3, 5C5B10, 13A12G4H2, 9C6B4, 6D9D10, 1F10A6 were thus obtained.
  • Table 1 summarizes the results obtained (RFV signal), with the different combinations of antibodies used in capture or detection, on three dilutions of the purified native annexin A3 neutrophil (100, 25 and 3 ng / mL ).
  • Table 1 Biotinylated revelation antibody Capture antibodies clone Dilution ANXA3 ng / ml TGC42 TGC43 TGC44 9C6B4 TGC42 0 - 22 11 17 3 - 44 20 78 25 - 238 89 500 100 - 859 297 1837 TGC43 0 106 - 39 63 3 111 - 36 82 25 144 - 56 257 100 232 - 86 914 TGC44 0 21 9 - 12 3 32 19 - 19 25 98 89 - 74 100 303 309 - 261 9C6B4 0 51 42 33 - 3 65 122 39 - 25 201 744 117 - 100 590 3525 357 - 5C5B10
  • the monoclonal antibodies TGC42, TGC43 and TGC44 have equivalent performances with respect to Annexin 3.
  • the monoclonal antibodies 5C5B10 and 13A12G4H2 also have equivalent performances with respect to Annexin 3, whereas the monoclonal antibody 1F10A6 gives satisfactory but weaker signals than with the monoclonal antibodies 5C5B10 and 13A12G4H2.
  • the complementarity and capacity of anti-ANXA3 antibodies to detect prostatic ANXA3 was analyzed using the ELISPOT technique.
  • This is a variant of the ELISA technique that directly detects secretions from cells in culture.
  • WPE1-NB26 human cells ATCC Cat No. CRL-2852
  • RWPE-1 prostatic epithelial line
  • MNU N-methyl-N-nitrosourea
  • the monoclonal capture antibodies (TGC42, TGC43, TGC44, 5C5B10, 13A12G4H2, 9C6D9, 1F10A6) were adsorbed on 96-well MultiScreen TM HTS plates (Millipore, Cat No. MSIP4510) at a concentration of 1 ⁇ g / well in sterile PBS. overnight at 4 ° C. The plates are then washed with PBS and saturated with culture medium containing 10% fetal calf serum (FCS). In parallel, the cells are counted, then diluted and distributed at 1000 cells per well. The plates are incubated for 20 hours at 37 ° C. and 5% CO 2 , and then emptied.
  • FCS fetal calf serum
  • the remaining cells are then lysed by treatment with ice water for 10 minutes.
  • the plates are then washed with PBS containing 0.05% Tween-20.
  • the biotinylated revelation antibodies (TGC42, TGC43, TGC44, 5C5B10, 13A12G4H2, 9C6D9, 1F10A6) are added at 0.1 ⁇ g / well diluted in PBS-1% BSA-0.05% Tween-20 and incubated for 2 hours at room temperature. room. After several washes, the spots are revealed by the addition of alkaline extravidine-phophatase (Sigma, Cat No. E2636) for one hour at room temperature. then bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium substrate (BCIP / NBT, Biorad, Cat No. 170-6432).
  • BCIP / NBT bromo-4-chloro-3-indolyl phosphate / nitro blue
  • the secretion of Annexin A3 by WPE1-NB26 cells is qualitatively measured by the observer.
  • the number of spots observed is plotted on a scale of - to ++++ (Table 3).
  • the best solution for detecting prostatic Annexin A3 is to match one of TGC42, TGC43 or TGC44 with an antibody selected from 13A12G4H2 and 1F10A6.
  • the selected antibodies can be used indifferently in capture or detection, the only condition is that one antibody is associated with each group.
  • the ANXA3 contained in these different biological samples was quantified with each of the nine selected ELISA assays.
  • the biotinylated detection antibodies were diluted to 0.5 ⁇ g / ml for the 5C5B10 and 13A12G4H2 monoclonals, to 1 ⁇ g / ml for the 1F10A6 clone.
  • a calibration curve was established by assaying a concentration range of the purified native Annexin A3 protein (Arodia). The calibration curve was plotted by plotting on the abscissa the logarithm in the base ten of the concentration and on the ordinate the signal measured by Vidas ® in RFV.
  • the concentration of ANXA3 present in the biological sample was calculated by interpolating the concentration corresponding to the RFV signal read by the Vidas®, using mathematical nonlinear regression models such as a third-order polynomial or a 4- model. PL, well known to those skilled in the art. The results are shown in Table 4 below.
  • the slope of the line calculated by placing on the abscissa the doses obtained by the ELISA using 13A12G4H2 and on the ordinate those obtained by the ELISA using 1F10A6 is 1: these two antibodies make identical doses.
  • the 9 ELISA assays can be divided into 2 groups.
  • Group 1 corresponds to the combinations of capture antibodies TGC42, TGC43 or TGC44 with clones 13A12G4H2 or 1F10A6 used in detection.
  • Group 2 corresponds to the combinations of capture antibodies TGC42, TGC43 or TGC44 with the 5C5B10 monoclonal used in detection. Since each of these antibodies is specific for ANXA3, both ELISA groups measure different biological information.
  • the ELISPOT assay presented above suggests that Group 1 ELISA using 13A12G4H2 or 1F10A6 clones are more suitable for detecting prostatic ANXA3.
  • TGC44 / 13A12G4H2 assay (called only 13A12G4H2 thereafter) was chosen as the assay Prototype Group 1 defined in Example 1.
  • TGC44 / 5C5B10 assay (called only 5C5B10 thereafter) was chosen as the prototype assay representing the group 2.
  • filtration and centrifugation fractionation results are extremely consistent and indicate that the majority of the ANXA3 to be assayed is associated with exosomes, but not only.
  • ANXA3 is also associated with larger particles such as cell debris, and that it can also be in soluble form.
  • the sample preparation for a Western blot analysis makes it possible to solubilize and denature the proteins and thus to reduce this complexity, which can partly explain the difficulties encountered so far in finding an ELISA assay method that can be used in the biological fluids and in particular urine.
  • the method of treatment of the expressed urine collected, the storage conditions are all factors that can vary the distribution of ANXA3 in the various urinary fractions where it may be present.
  • Annexin A3 contains in its protein sequence domains called annexin repeat. These repeated domains are four in number and characterize the family. To determine the repeated domain recognized by each of the monoclonal antibodies, these domains were expressed in a recombinant form. A sequence of 8 histidines was added to the N-terminal part of each domain to allow purification by metal-chelate affinity chromatography. Table 5 summarizes the protein sequences of the recombinant constructs making it possible to express each of the repeated domains in isolation.
  • the nucleic acid sequences corresponding to the protein sequences of the D1, D2, D3 and D4 domains were obtained by chemical synthesis by Geneart. These nucleic sequences have been optimized to promote the expression of proteins in Escherichia coli.
  • the DNA fragments were cloned between the Nco I and Xba I sites of the prokaryotic expression vector pMRCH79 (derived from pMR78, bioMérieux).
  • the plasmids thus obtained were transformed into BL21 (DE3) bacteria (Stratagene).
  • the cultures to produce the different domains are carried out at 37 ° C. with stirring in 2-YT medium (Invitrogen).
  • Each anti-ANXA3 antibody was tested with recombinants expressing the 4 repeated ANXA3 domains; the results of this Western blot analysis are presented in the Figure 5 .
  • the monoclonal antibodies TGC42, TGC43, TGC44 and 5C5B10 are specific for the D1 domain.
  • Monoclonal antibodies 13A12G4H2 and 1F10A6 are directed against the D4 domain.
  • the 9C6D4 and 6D9D10 antibodies they do not recognize any of the 4 repeat domains, which most likely indicates that their epitopes are located outside the repeated domains of ANXA3.
  • the epitopes deduced from the comparison of the sequences of the recognized overlapping peptides are summarized in Table 6.
  • the minimal epitope is the minimum sequence necessary to have a recognition of the antibody, with a more or less intense signal.
  • the optimal epitope is the ideal sequence for the best possible recognition of the antibody, including or identical to the minimal epitope.
  • Our results confirm that TGC42 and TGC43 are well directed against a single epitope, the one that is originally described in the application WO 2010/034825 .
  • the 5C5B10 antibody defines a new epitope that was not described in the prior art.
  • the antibodies 6D9D10 and 9C6D4 are specific for the N-terminus of the protein, as the monoclonal TGC7 of the application WO 2007/141043 .
  • the monoclonal antibodies TGC44, 13A12G4H2 and 1F10A6 show no Spotscan reactivity, even on a membrane carrying peptides 20 amino acids long. They probably have conformational or at least semi-conformational epitopes whose structures are not sufficiently well reproduced by synthetic peptides.
  • Table 6 Antibody Field Optimal epitope has Minimal epitope b TGC42 D1 SNAQRQLIVKEYQAAYG (49-65) (SEQ ID: 10) LIVKEYQAAYG (55-65) (SEQ ID: 11) TGC43 D1 IVKEYQAAYGKE (56-67) (SEQ ID: 12) KEYQAAYG (58-65) (SEQ ID: 13) 5C5B10 D1 DLSGHFEHL (75-83) (SEQ ID: 14) LSGHFEH (76-82) (SEQ ID: 15) 6D9D10 N-term ASIWVGHRGTVRDYPDFSPS (2-21) (SEQ ID: 16) SIWVGHRGTVRDYPDFSP (3-20) (SEQ ID: 17) 9C6D4 N-term YPDF (15-18) (SEQ ID: 18) YPDF (15-18) SEQ ID: 18) a Optimal epitope: Ideal sequence allowing the best possible recognition of the antibody (including or identical to the
  • the Novatope system (Merck, Cat No. 69279) is a technology for the analysis of a protein for the purpose of selecting domains containing epitopes.
  • the method is based on the creation of a library of bacterial clones, each expressing a fragment of the protein, cut randomly. These clones are analyzed by immunorevealing with the antibody that is to be characterized. DNA sequencing of the positive clones makes it possible to deduce the protein sequence from a fragment containing the epitope. The technique was applied following the procedure provided with the kit.
  • Clone 2J7 expresses the sequence KEYQAAYGKELKDDLKGDLSGHFEHLMVALVTPPAVFD (SEQ ID: 19) which corresponds to residues 58-95 of ANXA3.
  • Clone 2Z13 expresses the sequence QKAIRGIGTDEKMLISILTERSNAQRQLIVKEYQAAYGKELKDDLKGDLSGHFEHL (SEQ ID: 20) which corresponds to residues 28-83 of ANXA3.
  • the common part between the sequences of these two clones are the residues 58-83 of Annexin A3, that is the sequence KEYQAAYGKELKDDLKGDLSGHFEHL (SEQ ID: 21).
  • the TGC44 and 5C5B10 antibodies being complementary (see Example 1), their two epitopes can not be overlapping, one can thus remove the amino acids corresponding to the epitope of the antibody 5C5B10, or DLSGHFEHL (75-83) ( SEQ ID: 14).
  • the epitope of the TGC44 antibody is included in the sequence KEYQAAYGKELKDDLKG (58-74) (SEQ ID: 22). This is the same region as that recognized by TGC42 and TGC43.
  • the fact that the TGC44 antibody does not exhibit Spotscan reactivity indicates a conformational constraint for the recognition.
  • TGC44 is able to bind to its epitope only when the sequence KEYQAAYGKELKDDLKG (58-74) (SEQ ID: 22) is fused on the N-terminal side to a long sequence of at least 30 residues.
  • the recombinant repetitive domain D1 whose sequence is identified in SEQ ID NO: 6, clones 2J7 and 2Z13 fused to a carrier protein or the recombinant fragment vANA-7 described requires WO 2010/034825 are all recognized by clone TGC44.
  • the recombinant fragment vANA-3 which lacks the first 34 N-terminal amino acids, is not recognized ( Figure 6 )
  • the fold change signal is always around 0, indicating that none of the mutations tested disturb the binding of this monoclonal.
  • 13A12G4H2 and 1F10A6 it was possible to identify amino acids whose mutation prevents or disrupts the fixation very significantly. These are positions 260, 263, 265 and 306.
  • the mutation of position 270 has a measurable but less significant impact than for the previous positions. This shows that the amino acids 260, 263, 265 and 306 of ANXA3 belong to the epitope recognized by the monoclonal antibodies 13A12G4H2 and 1F10A6, the two most important residues in the antigen-antibody interaction being Lysine (K). position 263 and Aspartic Acid (D) at position 306.
  • Annexins are a family of proteins that share function and sequence homologies.
  • a BLAST query performed on the UniProtKB database, reduced to sequences of human origin, has made it possible to identify the proteins having the greatest sequence homology with Annexin A3. These are, in descending order of homology, annexin A4, annexin A11, annexin A6 and annexin A5.
  • the TGC44 / 13A12G4H2 assay (hereinafter called only 13A12G4H2) was chosen as the prototype group 1 assay defined in Example 1.
  • the TGC44 / 5C5B10 assay (hereinafter only referred to as 5C5B10) was chosen as the prototype assay representing group 2.
  • the lack of cross-reactivity was tested using commercially available antigens obtained from Abnova: annexin A1 (Cat No. H00000301-P01), Annexin A2 (Cat No.
  • Annexin A13 was expressed in recombinant form in the laboratory by cloning into the pMRCH79 expression vector; fused to a histidine tag, and then purified by metal-chelate affinity chromatography.
  • the proteins were diluted in the VIDAS well X1 buffer at a concentration of 12.5 ⁇ g / mL for TGC44 / 5C5B10 ELISA and at a concentration of 16 ⁇ g / mL for TGC44 / 13A12G4H2 ELISA.
  • the results are presented in the Figure 8 .
  • Both TGC44 / 13A12G4H2 and TGC44 / 5C5B10 ELISA formats are both highly specific for Annexin A3 and show no cross-reactivity with the other annexins tested.
  • the surface of the biosensor was regenerated by washing with 50 mM HCl at 10 ⁇ L / min for 120 seconds. The same measurement method was repeated for each dilution of the Annexin A3 protein; in total 9 different protein dilutions between 0 and 64 nM were analyzed. The resulting sensorgrams were plotted and analyzed with the dedicated Biacore TM T100 software, according to the 1: 1 interaction model.
  • the kinetic constants of association (Kon) and dissociation (Koff) were measured using the antibodies at a concentration of 3 ⁇ g / mL, except for 13A12G4H2 and 1F10A6 which were used at 0.75 ⁇ g / mL in order to limit the impact of background noise.
  • the set of anti-ANXA3 antibodies studied has high affinities, with Kd values ranging from 10 -9 to 5 x 10 -10 M.
  • the antibodies can be classified into two distinct groups. function of their Kd. The first group corresponds to antibodies 13A12G4H2, TGC42 and TGC44 whose Kd is greater than 10 -10 M, it is the group of very high affinity antibodies.
  • the second group corresponds to the antibodies 5C5B10, 1F10A6 and TGC43 whose Kd is between 10 -9 and 3.5 x 10 -9 M, it is the group of high affinity antibodies.
  • the 13A12G4H2 antibody also has a comparable association kinetic constant, thus confirming its capture capacity shown in Example 1.
  • the kinetic constant of the 5C5B10 antibody is approximately 1 log lower than that of TGC44. , which illustrates its lower catch capacity.
  • the kinetic dissociation constants it is also possible to divide the anti-ANXA3 antibodies into 2 groups.
  • the first group contains TGC44, TGC42, 13A12G4H2 and 5C5B10; it is characterized by a very low dissociation kinetic constant, between 9 x 10 -5 and 3.5 x 10 -4 s -1 .
  • Annexin A3 is retained by the antibodies in this group and does not dissociate.
  • the second group contains TGC43 and 1F10A6; it is characterized by a higher dissociation kinetic constant, of the order of 10 -3 s -1 . Antibodies in this group, even if they succeed in attaching Annexin A3, dissociate much more rapidly.
  • TGC42 and TGC44 are better capture antibodies to be used for the development of an ELISA assay than TGC43.
  • Table 7 Antibody fixed on the biosensor Kon (M -1 .s -1 ) Koff (s -1 ) Kd (M) 13A12G4H2 5,3E + 05 2.7E-04 5,0E-10 TGC42 7,3E + 05 1.0E-04 1,8E-10 TGC44 9,5E + 05 9,7E-05 1.0E-10 5C5B10 9,8E + 04 3.4E-04 3.4E-09 1F10A6 3,8E + 05 1,1E-03 2,9E-09 TGC43 8,4E + 05 1,1E-03 1.3E-09
  • Example 1 In order to analyze the ability of Annexin A3 to distinguish patients with prostate cancer from those who do not, the ELISA assays described in Example 1 were used to assay the amount of Annexin A3. present in the urine post-rectal examination of these patients.
  • the patients included in the "Cancer” group all have proven prostate cancer, whose diagnosis was confirmed by histological analysis on a biopsy.
  • prostate cancer was excluded by biopsy as well; the vast majority are patients with benign prostatic hyperplasia.
  • the collection and treatment of post-digital rectal examination urine samples were carried out according to the method described in Example 1.
  • Two cohorts of patients were analyzed: Cohort # 1 comprises 127 patients, 72 in the "Cancer” group and 55 in the "Non cancer” group.
  • Cohort # 2 includes 94 patients, 43 in the "Cancer” group and 42 in the "Non cancer” group. For both cohorts, these are patients with serum PSA between 2.5 and 10 ng / mL. This is the gray area of PSA, in which the clinical performance of the marker is the worst.
  • the first ELISA assays carried out on the urine expressed after the digital rectal examination, described in Example 1, made it possible to highlight a biological complexity that was previously unsuspected.
  • the TGC44 / 13A12G4H2 assay (hereinafter called 13A12G4H2 only) was chosen as the prototype group 1 assay defined in Example 1.
  • the TGC44 / 5C5B10 assay (referred to only as 5C5B10 thereafter) was chosen as a prototype assay representing group 2.
  • the Figure 10 represents the values obtained for each cohort # 1 and # 2 with both formats Annexin A3 ELISA assay used.
  • the two assay formats make it possible to discriminate cancer patients from controls.
  • the standardized doses of Annexin A3 are significantly lower in the "Cancer” group than in the "Non cancer” group, regardless of the dosage format used (unilateral Mann-Whitney p-value is 0.003 for the TGC44 / 5C5B10 assay and 0.02 for the TGC44 / 13A12G4H2 assay).
  • the second cohort it is only the TGC44 / 13A12G4H2 assay format that makes it possible to discriminate patients with cancer from controls.
  • cohort # 1 and in agreement with the Western blot study by Schostack et al.
  • the EDTA treatment of the urine has no effect on the 13A12G4H2 assay but for the 5C5B10 assay, it causes a very slight increase in the measured doses.

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Description

La présente invention a pour objet un procédé et un coffret pour le diagnostic in vitro du cancer de la prostate.The present invention relates to a method and a kit for the in vitro diagnosis of prostate cancer.

Le cancer de la prostate est le type de cancer le plus fréquent chez les hommes. Le cancer de la prostate a la particularité d'évoluer très lentement. Néanmoins, en dépit de son agressivité biologique modérée, comparée à d'autres types de cancers il s'agit du deuxième cancer le plus mortel chez les hommes, après le cancer du poumon, ex æquo avec le cancer colorectal (1). La gravité du cancer dépend de l'étendue de la tumeur (locale, avec métastases avoisinantes ou à distance) et du type de cellules cancéreuses, c'est-à-dire de leur degré de malignité. Il existe des solutions curatives, notamment pour les stades précoces de cancer, et limitées localement telle que la radiothérapie et/ou la chirurgie. Il est donc crucial de pouvoir disposer d'un test qui permet d'effectuer un diagnostic le plus précoce possible et fiable du cancer de la prostate.Prostate cancer is the most common type of cancer in men. Prostate cancer has the characteristic of evolving very slowly. Nevertheless, despite its moderate biological aggressiveness, compared to other types of cancer, it is the second most fatal cancer in men, after lung cancer, tied with colorectal cancer (1). The severity of the cancer depends on the extent of the tumor (local, with nearby or distant metastases) and the type of cancer cells, that is, the degree of malignancy. There are curative solutions, especially for early stages of cancer, and limited locally such as radiotherapy and / or surgery. It is therefore crucial to have a test that allows the earliest possible and reliable diagnosis of prostate cancer.

Le cancer de la prostate peut être dépisté par la constatation de l'augmentation de la concentration d'une protéine dans le sang, l'antigène prostatique spécifique ou PSA. Le PSA est produit par la prostate et est donc un marqueur spécifique de l'organe mais il n'est pas spécifique de la tumeur. C'est pourquoi un résultat élevé à un test de quantification de PSA ne signifie pas forcément qu'il y a un cancer. En effet, une quantité de plus de quatre nanogrammes par millilitre de cette protéine dans le sang est associée à un cancer de la prostate dans 25% des cas et à un autre trouble de la prostate dans 75% des cas, en particulier lors d'hypertrophie bénigne (BPH), d'une inflammation ou d'une infection de la prostate. De plus, bien qu'il soit relativement sensible pour les diagnostics initiaux et pour le suivi des patients, ce test ne permet pas dé détecter tous les cas de cancer de la prostate.Prostate cancer can be tracked by the finding of increased concentration of a protein in the blood, prostate specific antigen or PSA. PSA is produced by the prostate and is therefore a specific marker of the organ but it is not specific to the tumor. This is why a high result in a PSA quantification test does not necessarily mean that there is cancer. In fact, an amount of more than four nanograms per milliliter of this protein in the blood is associated with prostate cancer in 25% of cases and another prostate disorder in 75% of cases, especially when benign hypertrophy (BPH), inflammation or infection of the prostate. In addition, although it is relatively sensitive for initial diagnoses and for patient follow-up, this test does not detect all cases of prostate cancer.

Plus récemment, un autre marqueur a été décrit comme étant significativement associé à la progression et aux divers stades de maladies prostatiques et de cancer de la prostate. Il s'agit de l'Annexine 3 (ANXA3) qui appartient à la famille des Annexines. Les protéines de la famille des Annexines possèdent la caractéristique commune de pouvoir se fixer aux membranes phospholipidiques en présence de calcium. Pour ce faire, les membres de cette famille de protéines partagent des similitudes structurelles. Ainsi, chaque Annexine contient dans sa séquence protéique des domaines appelés « annexin repeat », qui sont au nombre de 4 ou 8 (4 pour l'Annexine A3), et ont une longueur d'environ 70 acides aminés (2). Chaque domaine répété, appelé aussi domaine endonexine, est replié en 5 hélices alpha et contient un motif caractéristique de type 2 (GxGT-[38 residus]-D/E) qui permet de fixer des ions Ca2+. Les annexines du règne animal possèdent des domaines N-terminaux variables, qui portent souvent les spécificités fonctionnelles des protéines. Les analyses cristallographiques montrent que les structures secondaires et tertiaires des annexines sont conservées même si l'identité des séquences en acides aminés ne dépasse pas 45 à 55%. Les 4 domaines répétés forment une structure qui ressemble à un disque, avec une surface un peu convexe sur laquelle se situent les sites de fixation du Ca2+ et une surface concave où les extrémités N- et C-terminales de la protéine se trouvent à proximité.More recently, another marker has been reported to be significantly associated with progression and various stages of prostate and prostate cancer. This is Annexin 3 (ANXA3) which belongs to the Annexines family. The The proteins of the Annexin family have the common characteristic of being able to bind to phospholipid membranes in the presence of calcium. To do this, the members of this protein family share structural similarities. Thus, each Annexin contains in its protein sequence domains called "annexin repeat", which are 4 or 8 (4 for Annexin A3), and have a length of about 70 amino acids (2). Each repetitive domain, also referred to as the endonexin domain, is folded into 5 alpha helices and contains a characteristic motif of type 2 (GxGT- [38 residues] -D / E) which makes it possible to fix Ca2 + ions. Annexins of the animal kingdom have variable N-terminal domains, which often carry the functional specificities of proteins. The crystallographic analyzes show that the secondary and tertiary structures of annexins are conserved even if the identity of the amino acid sequences does not exceed 45 to 55%. The 4 repeat domains form a disk-like structure with a slightly convex surface on which the Ca 2+ binding sites are located and a concave surface where the N- and C-terminal ends of the protein are found. proximity.

L'Annexine A3 est une annexine rare, qui n'est exprimée que faiblement ou pas du tout par la majorité des types cellulaires. A l'inverse, elle est très abondante dans les neutrophiles humaines, où elle représente environ 1% des protéines cytosoliques (3, 4). Initialement, deux isoformes de l'Annexine A3 ont été décrites, une de 33 kDa de masse moléculaire apparente, présente majoritairement dans les neutrophiles, l'autre de 36 kDa de masse moléculaire apparente, retrouvée dans les monocytes. La différence de masse moléculaire entre les deux isoformes ne résulte pas d'une modification post-traductionnelle telle que la phosphorylation ou la glycosylation. Très récemment, en 2010, il a été démontré qu'un phénomène d'épissage alternatif pouvait être un mécanisme expliquant la présence de ces deux isoformes. Ainsi, les auteurs ont identifié deux variants d'ADN complémentaire de l'Annexine A3 : le premier d'une longueur de 973 paires de bases (pb) code pour une protéine entière de 323 acides aminés correspondant à l'isoforme de 36 kDa ; le second d'une longueur de 885 pb ne contient pas l'exon III suite à un épissage alternatif. Lorsque cet exon n'est pas présent, le cadre de lecture ouvert est interrompu, et la traduction ne devient possible qu'à partir du codon ATG présent dans l'exon IV. Par conséquent, les 39 premiers acides aminés de la protéine ne sont pas exprimés, il en résulte une protéine tronquée à l'extrémité N-terminale de 284 acides aminés, correspondant à l'isoforme de 33 kDa (5).Annexin A3 is a rare annexin, which is expressed only slightly or not at all by the majority of cell types. Conversely, it is very abundant in human neutrophils, where it accounts for about 1% of cytosolic proteins (3, 4). Initially, two isoforms of Annexin A3 have been described, one of 33 kDa of apparent molecular weight, predominantly present in neutrophils, the other of 36 kDa apparent molecular weight, found in monocytes. The difference in molecular weight between the two isoforms does not result from a post-translational modification such as phosphorylation or glycosylation. Very recently, in 2010, it was shown that an alternative splicing phenomenon could be a mechanism for explaining the presence of these two isoforms. Thus, the authors identified two DNA variants complementary to Annexin A3: the first of 973 base pairs (bp) length encodes an entire protein of 323 amino acids corresponding to the 36 kDa isoform; the second with a length of 885 bp does not contain exon III following alternative splicing. When this exon is not present, the open reading frame is interrupted, and the translation becomes possible only from the ATG codon present in exon IV. As a result, the first 39 amino acids of the protein are not expressed, resulting in an N-terminal truncated protein of 284 amino acids, corresponding to the 33 kDa isoform (5).

De plus, l'analyse par Western blot après une électrophorèse à 2 dimensions (2DE-WB) montre que l'isoforme de 36 kDa migre sous la forme de 4 à 6 spots, ayant des points isoélectriques différents. L'isoforme de 36 kDa identifiée par un Western blot à 1 dimension correspond en fait à un groupe d'isoformes hétérogènes (5). C'est l'Annexine A3 de 36 kDa dont le niveau d'expression baisse en cas de cancer de la prostate (5-7).In addition, Western blot analysis after 2-dimensional electrophoresis (2DE-WB) shows that the 36 kDa isoform migrates as 4 to 6 spots, having different isoelectric points. The 36 kDa isoform identified by a 1-dimensional Western blot corresponds to a group of heterogeneous isoforms (5). It is the 36 kDa Annexine A3 whose level of expression decreases in case of prostate cancer (5-7).

Bien que l'Annexine A3 soit reconnue comme un marqueur pour le cancer de la prostate, les procédés pour la détecter décrits jusqu'à présent soit manquent de spécificité parce que les anticorps utilisés présentent des réactions croisées avec d'autres annexines ( WO 2006/125580 ), soit manquent de sensibilité pour la détecter dans un milieu complexe comme l'urine ( WO 2007/141043 ). De manière générale, les procédés antérieurs manquent de robustesse.Although Annexin A3 is recognized as a marker for prostate cancer, the methods for detecting it described so far are lacking in specificity because the antibodies used cross-react with other annexins ( WO 2006/125580 ), or lack sensitivity to detect it in a complex environment such as urine ( WO 2007/141043 ). In general, the prior methods lack robustness.

De manière surprenante, les inventeurs ont trouvé que pour pallier les inconvénients précités, il fallait utiliser un couple d'anticorps dont l'un est dirigé contre le premier domaine répété de l'Annexine A3 humaine native dont la séquence est identifiée en SEQ ID NO : 1 (acides aminés 27-87 de l'Annexine A3, selon la numérotation de la base de données UniProtKB de l'ANXA3 complète humaine n° d'accession P12429) et l'autre est dirigé contre le quatrième domaine répété de l'Annexine A3 humaine native dont la séquence est identifiée en SEQ ID NO : 2 (acides aminés 258-318, selon la numérotation de la base de données UniProtKB de l'ANXA3 complète humaine n° d'accession P12429).Surprisingly, the inventors have found that, to overcome the aforementioned drawbacks, it was necessary to use a pair of antibodies, one of which is directed against the first repeated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO. : 1 (amino acids 27-87 of Annexin A3, according to the numbering of the UniProtKB database of the complete ANXA3 Human Accession No. P12429) and the other is directed against the fourth repeated domain of the Native human Annexin A3 whose sequence is identified in SEQ ID NO: 2 (amino acids 258-318, according to the numbering of the UniProtKB database of the complete human ANXA3 Accession No. P12429).

Dans la description qui suit, la séquence du premier domaine répété de l'Annexine A3 humaine native est identifiée en SEQ ID NO : 1, la séquence du quatrième domaine répété de l'Annexine A3 humaine native est identifiée en SEQ ID NO : 2, la séquence du deuxième domaine répété de l'Annexine A3 humaine native est identifiée en SEQ ID NO : 3, la séquence du troisième domaine répété de l'Annexine A3 humaine native est identifiée en SEQ ID NO : 4. La séquence identifiée en SEQ ID NO : 5 correspond à la séquence en acides aminés de l'Annexine A3 humaine native complète (UniProtKB n° d'accession P12429).In the description which follows, the sequence of the first repeated domain of native human Annexin A3 is identified in SEQ ID NO: 1, the sequence of the fourth repeated domain of Annexin A3. native human is identified in SEQ ID NO: 2, the sequence of the second repeating domain of native human Annexin A3 is identified in SEQ ID NO: 3, the sequence of the third repeating domain of native human Annexin A3 is identified in SEQ ID NO: 4. The sequence identified in SEQ ID NO: 5 corresponds to the amino acid sequence of complete native human Annexin A3 (UniProtKB Accession No. P12429).

Aussi, la présente invention a pour objet un procédé pour le diagnostic in vitro d'un cancer de la prostate selon lequel on met en contact un échantillon d'urine à analyser avec deux anticorps, un anticorps de capture et un anticorps de détection, un des deux anticorps est dirigé contre le premier domaine répété de l'Annexine A3 humaine native dont la séquence est identifiée en SEQ ID NO : 1 et l'autre des deux anticorps est dirigé contre le quatrième domaine répété de l'Annexine A3 humaine native dont la séquence est identifiée en SEQ ID NO : 2.The subject of the present invention is therefore a method for the in vitro diagnosis of prostate cancer according to which a urine sample to be analyzed is brought into contact with two antibodies, a capture antibody and a detection antibody, a of the two antibodies is directed against the first repeated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 1 and the other of the two antibodies is directed against the fourth replicated domain of native human Annexin A3 whose the sequence is identified in SEQ ID NO: 2.

En particulier, l'anticorps dirigé contre le premier domaine répété de l'Annexine A3 humaine native est choisi parmi les anticorps dirigés contre un épitope dont la séquence en acides aminés comprend au moins 7 acides aminés consécutifs, de préférence au moins 8 ou au moins 9 acides aminés ou encore au moins 12 acides aminés consécutifs et pas plus que 17 acides aminés consécutifs de SEQ ID NO :1, et parmi ceux-ci on peut citer, à titre préférentiel, les anticorps qui sont dirigés contre un polypeptide inclus dans SEQ ID NO : 1 dont la séquence en acides aminés est sélectionnée parmi les séquences suivantes :

  • SNAQRQLIVKEYQAAYG (SEQ ID NO : 10),
  • LIVKEYQAAYG (SEQ ID NO : 11)
  • IVKEYQAAYGKE (SEQ ID NO : 12),
  • KEYQAAYG (SEQ ID NO : 13),
  • DLSGHFEHL (SEQ ID NO : 14),
  • LSGHFEH (SEQ ID NO : 15),
  • et
  • KEYQAAYGKELKDDLKG (SEQ ID NO : 22) à la condition que la séquence en acides aminés SEQ ID NO : 22 soit fusionnée du côté N-terminal à une séquence d'au moins 30 acides aminés.
In particular, the antibody directed against the first repeated domain of native human Annexin A3 is chosen from the antibodies directed against an epitope whose amino acid sequence comprises at least 7 consecutive amino acids, preferably at least 8 or at least 9 amino acids or at least 12 consecutive amino acids and not more than 17 consecutive amino acids of SEQ ID NO: 1, and among these there may be mentioned, preferably, the antibodies which are directed against a polypeptide included in SEQ ID NO: 1 whose amino acid sequence is selected from the following sequences:
  • SNAQRQLIVKEYQAAYG (SEQ ID NO: 10),
  • LIVKEYQAAYG (SEQ ID NO: 11)
  • IVKEYQAAYGKE (SEQ ID NO: 12),
  • KEYQAAYG (SEQ ID NO: 13),
  • DLSGHFEHL (SEQ ID NO: 14),
  • LSGHFEH (SEQ ID NO: 15),
  • and
  • KEYQAAYGKELKDDLKG (SEQ ID NO: 22) provided that the amino acid sequence SEQ ID NO: 22 is fused on the N-terminal side to a sequence of at least 30 amino acids.

Parmi les anticorps qui sont dirigés contre le quatrième domaine répété de l'Annexine A3 humaine native, on peut citer les anticorps qui sont dirigés contre un épitope dont la séquence en acides aminés dont la séquence en acides aminés comprend au moins 7 acides aminés consécutifs et pas plus que 50, de préférence pas plus que 45 acides aminés consécutifs de SEQ ID NO :2.Among the antibodies which are directed against the fourth repeating domain of native human Annexin A3, there may be mentioned antibodies which are directed against an epitope whose amino acid sequence whose amino acid sequence comprises at least 7 consecutive amino acids and not more than 50, preferably no more than 45 consecutive amino acids of SEQ ID NO: 2.

En particulier, les anticorps qui sont dirigés contre le quatrième domaine répété de l'Annexine A3 humaine native sont choisis parmi les anticorps qui sont dirigés contre un épitope qui :

  • est inclus dans une séquence en acides aminés correspondant à la séquence en acides aminés commençant au résidu 3 et se terminant au résidu 49 de SEQ ID NO : 2,
  • comprend en position 6 de SEQ ID NO : 2 un résidu Lys (K),
  • comprend en position 6 de SEQ ID NO : 2 un résidu Lys (K) et en position 49 de SEQ ID NO : 2 un résidu Asp (D),
  • comprend en position 7 de SEQ ID NO : 2 un résidu Gly (G), en position 8 de SEQ ID NO : 2 un résidu Ile (I) et en position 9 de SES ID NO : 2 un résidu Gly (G),
  • comprend en position 3 de SEQ ID NO : 2 un résidu Arg (R), en position 6 des SEQ ID NO : 2 un résidu Lys (K), en position 7 de SEQ ID NO : 2 un résidu Gly (G), en position 8 de SEQ ID NO : 2 un résidu Ile (I), en position 9 de SEQ ID NO : 2 un résidu Gly (G) et en position 49 de SEQ ID NO : 2 un résidu Asp (D).
In particular, the antibodies which are directed against the fourth repeating domain of native human Annexin A3 are selected from the antibodies which are directed against an epitope which:
  • is included in an amino acid sequence corresponding to the amino acid sequence beginning at residue 3 and ending at residue 49 of SEQ ID NO: 2,
  • comprises at position 6 of SEQ ID NO: 2 a residue Lys (K),
  • comprises at position 6 of SEQ ID NO: 2 a Lys (K) residue and at position 49 of SEQ ID NO: 2 an Asp residue (D),
  • comprises at position 7 of SEQ ID NO: 2 a Gly residue (G) at position 8 of SEQ ID NO: 2 an Ile residue (I) and at position 9 of SES ID NO: 2 a Gly residue (G),
  • comprises at position 3 of SEQ ID NO: 2 an Arg (R) residue at position 6 of SEQ ID NO: 2 a Lys (K) residue at position 7 of SEQ ID NO: 2 a Gly (G) residue, in position 8 of SEQ ID NO: 2 an Ile (I) residue, in position 9 of SEQ ID NO: 2 a Gly (G) residue and in position 49 of SEQ ID NO: 2 an Asp (D) residue.

Les positions 6, 49, 7, 8 et 9 déterminées par rapport à SEQ ID NO : 2 correspondent respectivement aux positions 263, 306, 264, 265 et 266 de l'Annexine A3 humaine identifiée en SEQ ID NO : 5 qui correspond à la séquence en acides aminés de l'Annexine A3 humaine native complète (UniProtKB n° d'accession P12429).The positions 6, 49, 7, 8 and 9 determined with respect to SEQ ID NO: 2 correspond respectively to the positions 263, 306, 264, 265 and 266 of the human Annexin A3 identified in SEQ ID NO: 5 which corresponds to the amino acid sequence of complete native Human Annexin A3 (UniProtKB Accession No. P12429).

De préférence, l'anticorps dirigé contre le premier domaine répétée de l'Annexine A3 humaine native, dont la séquence est identifiée en SEQ ID NO : 1, est l'anticorps de capture et l'anticorps dirigé contre le quatrième domaine répété de l'Annexine A3 humaine native, dont la séquence est identifiée en SEQ ID NO : 2, est l'anticorps de détection.Preferably, the antibody directed against the first repeated domain of native human Annexin A3, whose sequence is identified in SEQ ID NO: 1, is the capture antibody and the antibody directed against the fourth repetitive domain of the Native human Annexin A3, whose sequence is identified in SEQ ID NO: 2, is the detection antibody.

Les anticorps spécifiques du premier domaine répété de l'Annexine A3 native humaine et spécifiques du quatrième domaine répété de l'Annexine A3 native humaine, c'est à dire les anticorps de capture et de détection décrits ci-dessus, sont des anticorps qui présentent une affinité élevée avec une constante d'affinité d'au moins 10-9, de préférence d'au moins 10-10 et qui de plus présentent une constante de dissociation faible inférieure à 2 10-3 s-1, de préférence encore inférieure à 5 10-4 s-1.The antibodies specific for the first repeated domain of human native Annexin A3 and specific for the fourth repetitive domain of human native Annexin A3, that is to say the capture and detection antibodies described above, are antibodies which exhibit a high affinity with an affinity constant of at least 10 -9 , preferably at least 10 -10 and which furthermore have a low dissociation constant of less than 2 10 -3 s -1 , more preferably lower at 5 10 -4 s -1 .

De préférence les anticorps sont des anticorps monoclonaux et les anticorps préférés sont les anticorps suivants : TGC42, TGC43 et TGC44 utilisés comme anticorps de capture 13A12G4H2 et 1F10A6 utilisés comme anticorps de détection. Le couple préférentiel étant constitué par l'anticorps de capture TGC44 et l'anticorps de détection 13A12G4H2.Preferably the antibodies are monoclonal antibodies and the preferred antibodies are the following antibodies: TGC42, TGC43 and TGC44 used as capture antibodies 13A12G4H2 and 1F10A6 used as detection antibodies. The preferred pair consisting of the TGC44 capture antibody and the detection antibody 13A12G4H2.

L'invention a aussi pour objet un coffret d'immunodosage pour le diagnostic in vitro d'un cancer de la prostate dans un échantillon d'urine à analyser comprenant deux anticorps, un anticorps de capture et un anticorps de détection, un des deux anticorps étant dirigé contre le premier domaine répété de l'Annexine A3 humaine native dont la séquence est identifiée en SEQ ID NO : 1 et l'autre des deux anticorps anticorps étant dirigé contre le quatrième domaine répété de l'Annexine A3 humaine native dont la séquence est identifiée en SEQ ID NO : 2 et une notice explicative.The invention also relates to an immunoassay kit for the in vitro diagnosis of prostate cancer in an urine sample to be analyzed comprising two antibodies, a capture antibody and a detection antibody, one of the two antibodies. being directed against the first repeated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 1 and the other of the two antibody antibodies being directed against the fourth replicated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 2 and an explanatory note.

De préférence, l'anticorps de capture est dirigé contre le premier domaine répété de l'Annexine A3 humaine native dont la séquence est identifiée en SEQ ID NO : 1 et l'anticorps de détection dirigé contre le quatrième domaine répété de l'Annexine A3 humaine native dont la séquence est identifiée en SEQ ID NO : 2.Preferably, the capture antibody is directed against the first repeated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 1 and the detection antibody directed against the fourth repeated domain of Annexin A3. native human whose sequence is identified in SEQ ID NO: 2.

Les anticorps de capture et de détection du coffret présentent les mêmes caractéristiques que celles décrites ci-dessus pour le procédé.The capture and detection antibodies of the kit have the same characteristics as those described above for the process.

Par anticorps, on entend un anticorps polyclonal, un anticorps monoclonal, un anticorps humanisé, un anticorps humain ou un fragment desdits anticorps, en particulier les fragments Fab, Fab', F(ab')2, ScFv, Fv, Fd. La condition requise est que lesdits anticorps doivent être spécifiques de l'Annexine A3, c'est à dire qu'ils ne présentent de réactions croisées avec d'autres annexines et qu'ils sont spécifiques du premier domaine répété de l'Annexine A3 ou spécifiques du quatrième domaine répété de l'Annexine A3 ; les anticorps présentant les affinités les plus élevées et les constantes de dissociation les plus faibles étant les anticorps préférés.By antibody is meant a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody or a fragment of said antibodies, in particular Fab, Fab 'fragments, F (ab ') 2, ScFv, Fv, Fd. The requirement is that said antibodies must be specific for Annexin A3, that is to say that they do not cross-react with other annexins and that they are specific for the first repeated domain of Annexin A3 or specific to the fourth repeated domain of Annexin A3; the antibodies with the highest affinities and the weakest dissociation constants being the preferred antibodies.

Les anticorps polyclonaux peuvent être obtenus par immunisation d'un animal avec l'immunogène approprié, suivie de la récupération des anticorps recherchés sous forme purifiée, par prélèvement du sérum dudit animal, et séparation desdits anticorps des autres constituants du sérum, notamment par chromatographie d'affinité sur une colonne sur laquelle est fixé un antigène spécifiquement reconnu par les anticorps.The polyclonal antibodies can be obtained by immunizing an animal with the appropriate immunogen, followed by the recovery of the desired antibodies in purified form, by taking the serum of said animal, and separating said antibodies from the other constituents of the serum, in particular by chromatography. affinity on a column to which is attached an antigen specifically recognized by the antibodies.

Les anticorps monoclonaux peuvent être obtenus par la technique des hybridomes dont le principe général est rappelé ci-dessous.The monoclonal antibodies can be obtained by the hybridoma technique whose general principle is recalled below.

Dans un premier temps, on immunise un animal, généralement une souris avec l'immunogène approprié, dont les lymphocytes B sont alors capables de produire des anticorps contre cet antigène. Ces lymphocytes producteurs d'anticorps sont ensuite fusionnés avec des cellules myélomateuses "immortelles" (murines dans l'exemple) pour donner lieu à des hybridomes. A partir du mélange hétérogène des cellules ainsi obtenu, on effectue alors une sélection des cellules capables de produire un anticorps particulier et de se multiplier indéfiniment. Chaque hybridome est multiplié sous la forme de clone, chacun conduisant à la production d'un anticorps monoclonal dont les propriétés de reconnaissance vis-à-vis de la protéine pourront être testées par exemple en ELISA, par immunotransfert (Western blot) en une ou deux dimensions, en immunofluorescence, ou à l'aide d'un biocapteur. Les anticorps monoclonaux ainsi sélectionnés, sont par la suite purifiés notamment selon la technique de chromatographie d'affinité décrite ci-dessus.Firstly, an animal, usually a mouse, is immunized with the appropriate immunogen whose B cells are then capable of producing antibodies against this antigen. These antibody-producing lymphocytes are then fused with "immortal" myeloma cells (murine in the example) to give rise to hybridomas. From the heterogeneous mixture of cells thus obtained, the cells capable of producing a particular antibody and of multiplying indefinitely are then selected. Each hybridoma is multiplied in the form of a clone, each leading to the production of a monoclonal antibody whose protein recognition properties can be tested, for example by ELISA, by immunoblot (Western blot) in one or two-dimensional, in immunofluorescence, or with a biosensor. The monoclonal antibodies thus selected are subsequently purified, in particular according to the affinity chromatography technique described above.

Les anticorps monoclonaux peuvent être également des anticorps recombinants obtenus par génie génétique, par des techniques bien connues de l'homme du métier.The monoclonal antibodies can also be recombinant antibodies obtained by genetic engineering, by techniques well known to those skilled in the art.

L'anticorps de capture est de préférence fixé, directement ou indirectement, sur un support solide, par exemple un cône, un puits d'une plaque de microtitration etc...The capture antibody is preferably fixed, directly or indirectly, on a solid support, for example a cone, a well a microtitration plate etc ...

L'anticorps de détection est marqué à l'aide d'un réactif marqueur capable de générer directement ou indirectement un signal détectable. Une liste non limitative de ces réactifs marqueurs consiste en :

  • les enzymes qui produisent un signal détectable par exemple par colorimétrie, fluorescence, luminescence, comme la peroxydase de raifort, la phosphatase alcaline, la β-galactosidase, la glucose-6-phosphate déshydrogénase,
  • les chromophores comme les composés fluorescents, luminescents, colorants,
  • les molécules fluorescentes telles que les Alexa ou les phycocyanines,
  • les molécules radioactives comme le 32P, le 35S ou le 125I.
The detection antibody is labeled with a marker reagent capable of directly or indirectly generating a detectable signal. A nonlimiting list of these marker reagents consists of:
  • enzymes which produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase,
  • chromophores such as fluorescent compounds, luminescent compounds, dyes,
  • fluorescent molecules such as Alexa or phycocyanines,
  • radioactive molecules such as 32 P, 35 S or 125 I.

Des systèmes indirects de détection peuvent être aussi utilisés, comme par exemple des ligands capables de réagir avec un anti-ligand. Les couples ligand/anti-ligand sont bien connus de l'homme du métier, ce qui est le cas par exemple des couples suivants : biotine/streptavidine, haptène/anticorps, antigène/anticorps, peptide/anticorps, sucre/lectine. Dans ce cas, c'est le ligand qui porte le partenaire de liaison. L'anti-ligand peut être détectable directement par les réactifs marqueurs décrits au paragraphe précédent ou être lui-même détectable par un ligand/anti-ligand.Indirect detection systems can also be used, such as, for example, ligands capable of reacting with an anti-ligand. The ligand / anti-ligand couples are well known to those skilled in the art, which is the case, for example, of the following pairs: biotin / streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, sugar / lectin. In this case, it is the ligand that carries the binding partner. The anti-ligand can be detectable directly by the labeling reagents described in the preceding paragraph or be itself detectable by a ligand / anti-ligand.

Ces systèmes indirects de détection peuvent conduire, dans certaines conditions, à une amplification du signal. Cette technique d'amplification du signal est bien connue de l'homme du métier, et l'on pourra se reporter aux demandes de brevet antérieures FR2781802 ou WO-A-95/08000 de la Demanderesse.These indirect detection systems can lead, under certain conditions, to amplification of the signal. This signal amplification technique is well known to those skilled in the art, and reference can be made to prior patent applications. FR2781802 or WO-A-95/08000 of the Applicant.

Selon le type de marquage utilisé, l'homme du métier ajoutera des réactifs permettant la visualisation du marquage.Depending on the type of labeling used, those skilled in the art will add reagents allowing the visualization of the marking.

Le procédé de l'invention est un immunodosage de type « sandwich » effectué sur un prélèvement d'urine, en particulier un prélèvement d'urine « exprimée », c'est à dire un échantillon d'urine obtenu après toucher rectal.The method of the invention is a "sandwich" type immunoassay carried out on a urine sample, in particular an "expressed" urine sample, that is to say a urine sample obtained after digital rectal examination.

L'invention sera mieux comprise à l'aide des exemples suivants donnés à titre illustratif, ainsi qu'à l'aide des figures annexées.The invention will be better understood with the aid of the following examples given for illustrative purposes, as well as with the aid of the appended figures.

FIGURESFIGURES

  • La figure 1 correspond aux graphes relatifs au dosage ELISA de l'Annexine A3 en ng/mL, dans les urines exprimées après le toucher rectal, chez 6 patients (Patients A à F), dosées individuellement avec chacun des formats de dosage ELISA sandwich dont le nom est indiqué en abscisse. Les valeurs numériques représentées sur ce graphe sont présentées dans le tableau 4 ;The figure 1 corresponds to the graphs relating to the ELISA Annexin A3 assay in ng / mL, in the urine expressed after the digital rectal examination, in 6 patients (Patients A to F), individually assayed with each of the sandwich ELISA assay formats whose name is indicated on the abscissa. The numerical values represented on this graph are presented in Table 4;
  • la figure 2 représente le dosage ELISA de l'Annexine A3 lors de l'analyse par filtrations successives (sur filtre de 0,45 µm, puis de 0,22 µm, puis de 0,02 µm) de huit urines exprimées après le toucher rectal. L'analyse a été effectuée dans les 3-4 heures qui suivent la collecte de l'échantillon. Panel dosage 5C5B10 : les échantillons et leurs fractions ont été dosés en utilisant l'ELISA TGC44/5C5B10. Panel dosage 13A12G4H2 : les échantillons et leurs fractions ont été dosés en utilisant l'ELISA TGC44/13A12G4H2. NF : non filtré, contrôle ; FT : filtrat avec le seuil de coupure du filtre utilisé indiqué. Pour chaque groupe de 8 fractions la ligne horizontale matérialise la moyenne des 8 mesures observées. Les doses d'Annexine A3 sont exprimées en % de la dose de départ ;the figure 2 represents the ELISA Annexin A3 assay during the analysis by successive filtrations (on a filter of 0.45 μm, then of 0.22 μm, then of 0.02 μm) of eight urine expressed after the digital rectal examination. The analysis was performed within 3-4 hours after sample collection. 5C5B10 assay panel: The samples and their fractions were assayed using the TGC44 / 5C5B10 ELISA. 13A12G4H2 assay panel: The samples and their fractions were assayed using the TGC44 / 13A12G4H2 ELISA. NF: unfiltered, control; FT: filtrate with the cut-off point of the filter used indicated. For each group of 8 fractions, the horizontal line represents the average of the 8 measurements observed. The doses of Annexin A3 are expressed as a% of the starting dose;
  • la figure 3 représente le dosage ELISA de l'Annexine A3 lors de l'analyse par centrifugations successives (800 g, puis 12 000 g, 150 000 g) de huit urines exprimées après le toucher rectal. L'analyse a été effectuée dans les 3-4 heures qui suivent la collecte de l'échantillon. Panel dosage 5C5B10 : les échantillons et leurs fractions ont été dosés en utilisant l'ELISA TGC44/5C5B10. Panel dosage 13A12G4H2 : les échantillons et leurs fractions ont été dosés en utilisant l'ELISA TGC44/13A12G4H2. NC : non centrifugé, contrôle ; SN : surnageant avec la vitesse de centrifugation indiquée. Pour chaque groupe de 8 fractions la ligne horizontale matérialise la moyenne des 8 mesures observées. Les doses d'Annexine A3 sont exprimées en % de la dose de départ ;the figure 3 represents the ELISA assay of Annexin A3 during the analysis by successive centrifugations (800 g, then 12,000 g, 150,000 g) of eight urine expressed after digital rectal examination. The analysis was performed within 3-4 hours after sample collection. 5C5B10 assay panel: The samples and their fractions were assayed using the TGC44 / 5C5B10 ELISA. 13A12G4H2 assay panel: The samples and their fractions were assayed using the TGC44 / 13A12G4H2 ELISA. NC: non-centrifuged, control; SN: supernatant with the indicated centrifugation rate. For each group of 8 fractions, the horizontal line represents the average of the 8 measurements observed. The doses of Annexin A3 are expressed as a% of the starting dose;
  • la figure 4 représente le dosage ELISA de l'Annexine A3 lors de l'analyse par centrifugations successives (800 g, 12 000 g, 150 000 g) de dix autres urines exprimées après le toucher rectal. L'analyse a été effectuée dans les 3-4 heures qui suivent la collecte de l'échantillon. Panel dosage 5C5B10 : les échantillons et leurs fractions ont été dosés en utilisant l'ELISA TGC44/5C5B10. Panel dosage 13A12G4H2 : les échantillons et leurs fractions ont été dosés en utilisant l'ELISA TGC44/13A12G4H2. % soluble = dose dans surnageant d'ultracentrifugation / dose initiale x 100 ; % exosome = (surnageant 12 000 g / dose initiale x 100) - % soluble ; % membrane = 100 -(surnageant 12 000 g / dose initiale x 100).the figure 4 represents the ELISA assay of Annexin A3 in the successive centrifugation assay (800 g , 12,000 g, 150,000 g) of ten other urine expressed after digital rectal examination. The analysis was performed within 3-4 hours after sample collection. 5C5B10 assay panel: The samples and their fractions were assayed using the TGC44 / 5C5B10 ELISA. 13A12G4H2 assay panel: The samples and their fractions were assayed using the TGC44 / 13A12G4H2 ELISA. % soluble = dose in ultracentrifugation supernatant / initial dose x 100; % exosome = (supernatant 12,000 g / initial dose x 100) -% soluble; % membrane = 100 - (supernatant 12,000 g / initial dose x 100).
  • la figure 5 représente la réactivité des 4 domaines répétés D1 à D4 de l'Annexine A3, exprimés sous une forme recombinante, avec les 6 anticorps monoclonaux anti-Annexine A3 indiqués et analysée par la technique du Western blot. Pour les gels TGC42 et 1F10A6, le premier puits correspond au marqueur de poids moléculaire ;the figure 5 represents the reactivity of the 4 repeated D1 to D4 domains of Annexin A3, expressed in a recombinant form, with the 6 anti-Annexin A3 monoclonal antibodies indicated and analyzed by the Western blot technique. For the TGC42 and 1F10A6 gels, the first well corresponds to the molecular weight marker;
  • la figure 6 est un alignement des séquences des différentes protéines recombinantes exprimant des domaines d'Annexine A3 et récapitule l'immunoréactivité de chacun de ces recombinants avec l'anticorps monoclonal TGC44, analysée par la technique du Western blot. Les trois points de suspension à l'extrémité N-terminale ou C-terminale d'une séquence indiquent qu'elle continue dans la construction, même si elle n'est pas représentée en totalité sur la figure. Le codon stop est représenté par une étoile (*) ;the figure 6 is an alignment of the sequences of the different recombinant proteins expressing Annexin A3 domains and recapitulates the immunoreactivity of each of these recombinants with the monoclonal antibody TGC44, analyzed by the Western blot technique. The three points of suspension at the N-terminus or C-terminus of a sequence indicate that it continues in the construction, even though it is not shown entirely in the figure. The stop codon is represented by a star (*);
  • la figure 7 illustre l'impact des mutations Alanine du domaine D4 sur la reconnaissance de l'ANXA3 par les anticorps 13A12G4H2 et 1F10A6. L'analyse a été faite par la technique ELISA. L'anticorps de capture est le TGC44. Les anticorps 13A12G4H2, 1F10A6 testés ont été utilisés en détection. La position des mutations Alanine sur la séquence protéique de l'ANXA3 est représentée en abscisse. L'ordonnée est le « signal fold change » qui correspond au log2 (signal protéine mutée / signal protéine non mutée). L'anticorps 5C5B10 qui n'est pas dirigé contre le domaine D4 n'est pas impacté, il sert de contrôle. Les flèches indiquent les mutations pour lesquelles il y a perturbation du signal de reconnaissance pour 13A12G4H2 et 1F10A6, qui permettent de définir les résidus impliqués dans la fixation de ces anticorps.the figure 7 illustrates the impact of Alanine mutations of the D4 domain on the recognition of ANXA3 by the 13A12G4H2 and 1F10A6 antibodies. The analysis was done by the ELISA technique. The capture antibody is TGC44. The 13A12G4H2, 1F10A6 antibodies tested were used in detection. The position of Alanine mutations on the protein sequence of ANXA3 is represented as abscissa. The ordinate is the "fold change signal" which corresponds to log 2 (mutated protein signal / non-mutated protein signal). The 5C5B10 antibody that is not directed against the D4 domain is not impacted, it serves as a control. The arrows indicate the mutations for which there is a disturbance of the recognition signal for 13A12G4H2 and 1F10A6, which make it possible to define the residues involved in the binding of these antibodies.
  • la figure 8 illustre l'absence de réactivité croisée des formats de dosage ELISA décrits avec 7 autres protéines de la famille des annexines. Le graphique représente le signal ELISA obtenu en « Relative Fluorescence Values » (RFV) sur l'appareil VIDAS® pour chacun des formats de test TGC44/5C5B10 et TGC44/13A12G4H2 et pour chaque annexine indiquée en abscisse. A titre de comparaison, dans les conditions expérimentales utilisées, l'Annexine A3 permettait d'obtenir un signal supérieur à 6000 RFV avec chacun des formats de test ;the figure 8 illustrates the absence of cross-reactivity of the ELISA assay formats described with 7 other proteins of the annexin family. The graph represents the ELISA signal obtained in Relative Fluorescence Values (RFV) on the VIDAS® apparatus for each of the test formats TGC44 / 5C5B10 and TGC44 / 13A12G4H2 and for each annexin indicated on the abscissa. By way of comparison, under the experimental conditions used, Annexin A3 made it possible to obtain a signal greater than 6000 RFV with each of the test formats;
  • la figure 9 représente les sensorgrammes obtenus pour chacun des 6 anticorps monoclonaux caractérisés à l'aide du Biacore™ T100. Les graphiques représentent le signal de résonance mesuré en « Resonance Units » (RU) en fonction du temps. Chaque courbe d'un graphique représente l'ensemble des mesures effectuées pour une concentration d'Annexine A3 donnée. Pour chaque anticorps 9 dilutions comprises entre 0 et 64 nM d'Annexine A3 ont été analysées et sont représentées ;the figure 9 represents the sensorgrams obtained for each of the 6 monoclonal antibodies characterized using Biacore ™ T100. The graphs represent the resonance signal measured in "Resonance Units" (RU) as a function of time. Each curve in a graph represents all the measurements taken for a given Annexin A3 concentration. For each antibody 9 dilutions between 0 and 64 nM Annexin A3 were analyzed and are shown;
  • la figure 10 regroupe des graphes relatifs au dosage par ELISA de l'Annexine A3 dans les urines exprimées après le toucher rectal, chez des patients présentant un cancer de la prostate confirmé par biopsie (cancer), et des patients ayant une pathologie de la prostate mais dont la malignité a été exclue (pas cancer). Le groupe « pas cancer » est constitué majoritairement de patients ayant une hypertrophie bénigne de la prostate. Les graphes à la gauche de la page intitulés 5C5B10_SG représentent la dose d'Annexine A3 mesurée par le dosage VIDAS TGC44/5C5B10 en ng/mL et normalisée par la densité urinaire mesurée par la bandelette Combur™ 10 selon la formule : dose normalisée=dose VIDAS/(densité urinaire - 1). De la même manière, les graphes à la droite de la page intitulés 13A12G4H2_SG représentent la dose d'Annexine A3 mesurée par le dosage VIDAS TGC44/13A12G4H2 en ng/mL et normalisée par la densité urinaire. Deux différentes cohortes de patients ont été étudiées : la cohorte #1 comporte 127 patients et la cohorte #2 94 patients. Pour chaque série de données, la moyenne de la série est représentée sous forme d'un trait horizontal sur les graphes. La probabilité associée au test de Mann-Whitney unilatéral est également indiquée sur chaque graphe : p-value < 0,05 = *, p-value < 0,01 = **, ns = non significatif.the figure 10 groups together graphs relating to the ELISA Annexin A3 assay in urines expressed after digital rectal examination, in patients with biopsy-confirmed prostate cancer (cancer), and patients with prostate pathology but whose malignancy was excluded (not cancer). The group "not cancer" consists mainly of patients with benign prostatic hypertrophy. The graphs on the left of the page titled 5C5B10_SG represent the dose of Annexin A3 measured by the VIDAS TGC44 / 5C5B10 assay in ng / mL and normalized by the urinary density measured by the Combur ™ 10 strip according to the formula: normalized dose = dose VIDAS / (urinary density - 1). Similarly, the graphs on the right of the page labeled 13A12G4H2_SG represent the dose of Annexin A3 measured by the VIDAS TGC44 / 13A12G4H2 assay in ng / mL and normalized by urinary density. Two different cohorts of patients were studied: cohort # 1 has 127 patients and cohort # 2 has 94 patients. For each series of data, the series mean is represented as a horizontal line on the graphs. The probability associated with the unilateral Mann-Whitney test is also indicated on each graph: p-value < 0.05 = *, p-value <0.01 = **, ns = not significant.
  • la figure 11 représente l'effet de l'ion calcium et de l'agent de chélation EDTA sur les doses mesurées avec les dosages 5C5B10 et 13A12G4H2. Onze urines recueilles après le toucher rectal ont été dosées directement (sans traitement) ou après addition de 5 ou 25 mM de CaCl2 ou après addition de 5 ou 25 mM d'EDTA. L'axe des ordonnées représente le ratio des doses avec traitement (Ca2+ ou EDTA) / dose sans traitement. La médiane des séries est représentée sous forme d'un trait horizontal sur les graphes. La probabilité associée au test de Wilcoxon signed-rank bilatéral permettant de comparer l ratio à 1, cette probabilité ayant été corrigée pour tests multiples selon Bonferroni, est également indiquée sur chaque série.the figure 11 represents the effect of calcium ion and EDTA chelator on the doses measured with the 5C5B10 and 13A12G4H2 assays. Eleven urine collected after digital rectal examination were dosed directly (without treatment) or after addition of 5 or 25 mM CaCl 2 or after addition of 5 or 25 mM EDTA. The y-axis represents the ratio of the doses with treatment (Ca 2+ or EDTA) / dose without treatment. The median of the series is represented as a horizontal line on the graphs. The probability associated with the Wilcoxon signed-rank test comparing the ratio to 1, this probability having been corrected for multiple tests according to Bonferroni, is also indicated on each series.
EXEMPLESEXAMPLES Exemple 1 : Détection de l'Annexine A3 par ELISA dans différents types d'échantillonsExample 1: Detection of Annexin A3 by ELISA in Different Types of Samples Obtention des anticorps monoclonauxObtaining monoclonal antibodies

Les expériences d'immunisations ont été réalisées chez des souris BALB/c (H-2d) femelles âgées de six à huit semaines au moment de la première immunisation. La protéine Annexine A3 native humaine a été achetée auprès de la société Arodia Arotech Diagnostic (Cat No. 25592), elle est purifiée à partir de neutrophiles humaines. Cette protéine a été mélangée volume pour volume avec l'adjuvant de Freund (Sigma), préparé sous forme d'émulsion eau-dans-huile et dont il est connu qu'il présente un bon pouvoir immunogène. Elles ont reçu trois doses successives de 10 µg d'immunogène à zéro, deux et quatre semaines. Toutes les injections sont réalisées par voie sous cutanée. La première injection est faite en mélange avec l'adjuvant de Freund complet, les deux suivantes se font en mélange avec l'adjuvant de Freund incomplet. Entre J50 et J70 après la première injection , les réponses humorales ont été restimulées avec une injection intraveineuse de 100 µg de la protéine native.Immunization experiments were performed in female BALB / c (H-2 d ) mice six to eight weeks old at the time of the first immunization. Native human Annexin3 protein was purchased from Arodia Arotech Diagnostic (Cat No. 25592) and is purified from human neutrophils. This protein was mixed volume for volume with Freund's adjuvant (Sigma), prepared as a water-in-oil emulsion and which is known to have good immunogenicity. They received three successive doses of 10 μg of immunogen at zero, two and four weeks. All injections are performed subcutaneously. The first injection is made in admixture with the complete Freund's adjuvant, the next two are mixed with the incomplete Freund's adjuvant. Between D50 and D70 after the first injection, the humoral responses were restimulated with an intravenous injection of 100 μg of the native protein.

Afin de suivre l'apparition des anticorps, on effectue régulièrement sur les souris des prélèvements de sang. La présence des anticorps anti-ANXA3 est testée en utilisant un ELISA. La protéine d'intérêt est utilisée en capture (1 µg/puit), après saturation on fait réagir avec l'antigène différentes dilutions de sérums à tester (incubation à 37°C, pendant 1h). Les anticorps spécifiques présents dans le sérum sont révélés par un anticorps de chèvre anti-IgG de souris AffiniPure conjugué à la phosphatase alcaline (H+L, Jackson Immunoresearch, Cat no. 115-055-146), qui se lie aux anticorps recherchés (0.1µg/puit).In order to follow the appearance of the antibodies, blood samples are regularly taken from the mice. The presence of anti-ANXA3 antibodies is tested using an ELISA. The protein of interest is used in capture (1 μg / well), after saturation antigen is reacted with different dilutions of test sera (incubation at 37 ° C for 1h). The specific antibodies present in the serum are revealed by a goat anti-mouse IgG AffiniPure conjugated to phosphatase alkaline (H + L, Jackson Immunoresearch, Cat No. 115-055-146), which binds to the desired antibodies (0.1 μg / well).

Trois jours après la dernière injection, une des souris immunisée a été sacrifiée ; le sang et la rate ont été prélevés. Les splénocytes obtenues à partir de la rate ont été mises en culture avec les cellules de myélome Sp2/0-Ag14 pour qu'elles fusionnent et s'immortalisent, selon le protocole décrit par (8, 9). Après une période d'incubation de 12-14 jours les surnageants d'hybridomes obtenus ont été criblés pour déterminer la présence d'anticorps anti-ANXA3 en utilisant le test ELISA décrit dans le paragraphe précédent. L'immunogène (ANXA3 native), l'Annexine A3 recombinante produite en E. coli, et différentes cellules humaines exprimant l'ANXA3 ont été utilisés successivement pour cribler les surnageants d'hybridomes. Les colonies d'hybridomes positives ont été sous-clonées deux fois selon la technique de la dilution limite, bien connue par l'homme du métier.Three days after the last injection, one of the immunized mice was sacrificed; blood and spleen were removed. Splenocytes obtained from the spleen were cultured with Sp2 / 0-Ag14 myeloma cells to fuse and immortalize, according to the protocol described by (8, 9). After a 12-14 day incubation period, the resulting hybridoma supernatants were screened for the presence of anti-ANXA3 antibodies using the ELISA test described in the previous paragraph. The immunogen (native ANXA3), recombinant Annexin A3 produced in E. coli, and various human cells expressing ANXA3 were used successively to screen the hybridoma supernatants. Positive hybridoma colonies were subcloned twice according to the limiting dilution technique, well known to those skilled in the art.

Les anticorps monoclonaux anti-annexine A3, 5C5B10, 13A12G4H2, 9C6B4, 6D9D10, 1F10A6 ont ainsi été obtenus.The anti-annexin monoclonal antibodies A3, 5C5B10, 13A12G4H2, 9C6B4, 6D9D10, 1F10A6 were thus obtained.

Sélection des anticorps monoclonaux anti-ANXA3 pour dosage par immunoessai de l'ANXA3Selection of anti-ANXA3 monoclonal antibodies for immunoassay assay of ANXA3

La complémentarité des différents anticorps anti-ANXA3 obtenus comme décrit ci-dessus et des anticorps TGC42, TGC43 et TGC44 décrits dans la demande de brevet WO 2010/034825 a été analysé en utilisant comme antigène l'ANXA3 native (immunogène) dilué en tampon PBS, en réalisant en immunoessai de type sandwich. Ce type de dosage peut être réalisé en microplaque, de façon automatisée ou manuelle, ou encore en utilisant des automates d'immunoanalyse comme le VIDAS (bioMérieux).
Nous avons utilisé les réactifs du kit Vidas® HBs Ag Ultra (bioMérieux, Cat no 30315), tels que décrits dans la notice correspondante (réf. 11728 D- FR-2005/5 ) et modifiée ainsi :

  • Des cônes ont été sensibilisés avec un des anticorps de capture à tester, TGC42, TGC43, TGC44 ou 9C6B4 à une concentration de 10 µg /mL.
  • Le contenu du deuxième puits de la cartouche HBs Ag Ultra a été remplacé par 300 µL d'anticorps de révélation à tester (5C5B10, 13A12G4H2, 9C6B4, 6D9D10, 1F10A6, TGC42, TGC43, TGC44), couplés à la biotine, dilués à 1µg/mL dans le tampon du deuxième puits du kit Vidas® HBs Ag Ultra (puits X1) contenant du sérum de chèvre et de l'azodure de sodium à 1g/L.
  • La protéine ANXA3 native est testée diluée à 100, 25 et 3 ng/mL dans du tampon PBS. L'échantillon est déposé (150 µL) dans le premier puits (puits X0) de la cartouche HBs Ag Ultra.
  • La réaction ELISA a été réalisée à l'aide de l'automate Vidas® et du protocole décrit pour le test HBs Ag Ultra.
  • Les résultats ont été obtenus sous formes de valeurs brutes après soustraction du bruit de fond. Le signal est en RFV (relative fluorescente value).
The complementarity of the different anti-ANXA3 antibodies obtained as described above and the TGC42, TGC43 and TGC44 antibodies described in the patent application WO 2010/034825 was analyzed using as antigen the native ANXA3 (immunogenic) diluted in PBS buffer, performing in sandwich immunoassay. This type of assay can be performed in microplate, automated or manual, or by using automated immunoassays such as VIDAS (bioMérieux).
We used the reagents of the Vidas® HBs Ag Ultra Kit (bioMérieux, Cat No. 30315), as described in the corresponding leaflet (Cat. EN-2005/5 ) and amended as follows:
  • Cones were sensitized with one of the capture antibodies to be tested, TGC42, TGC43, TGC44 or 9C6B4 at a concentration of 10 μg / mL.
  • The content of the second well of the HBs Ag Ultra cartridge was replaced by 300 μL of test revealing antibody (5C5B10, 13A12G4H2, 9C6B4, 6D9D10, 1F10A6, TGC42, TGC43, TGC44), coupled to biotin, diluted to 1 μg / ml in the buffer of the second well of the Vidas® HBs Ag Ultra Kit (well X1) containing goat serum and sodium azodide at 1 g / L.
  • The native ANXA3 protein is tested diluted at 100, 25 and 3 ng / mL in PBS buffer. The sample is deposited (150 μL) in the first well (well X0) of the HBs Ag Ultra cartridge.
  • The ELISA reaction was carried out using the Vidas® automaton and the protocol described for the HBs Ag Ultra test.
  • The results were obtained as raw values after subtraction of the background noise. The signal is in RFV (relative fluorescent value).

Le tableau 1 ci-dessous résume les résultats obtenus (signal RFV), avec les différentes combinaisons d'anticorps utilisés en capture ou en détection, sur trois dilutions de l'annexine A3 native purifiée des neutrophiles (100, 25 et 3 ng/mL). Tableau 1 Anticorps de révélation biotinylés Anticorps de capture Clone Dilution ANXA3 ng/ml TGC42 TGC43 TGC44 9C6B4 TGC42 0 - 22 11 17 3 - 44 20 78 25 - 238 89 500 100 - 859 297 1837 TGC43 0 106 - 39 63 3 111 - 36 82 25 144 - 56 257 100 232 - 86 914 TGC44 0 21 9 - 12 3 32 19 - 19 25 98 89 - 74 100 303 309 - 261 9C6B4 0 51 42 33 - 3 65 122 39 - 25 201 744 117 - 100 590 3525 357 - 5C5B10 0 20 12 93 9 3 5244 3669 5870 39 25 10480 9874 10429 249 100 11310 11095 11168 1139 13A12G4H2 0 26 21 18 19 3 2298 676 3733 23 25 9655 8468 10208 21 100 11380 10995 11332 21 6D9D10 0 12 23 16 9 3 11 23 14 9 25 12 23 18 19 100 18 41 43 27 1F10A6 0 45 9 14 14 3 159 207 194 17 25 4014 6728 5680 13 100 10489 10931 10688 15 Table 1 below summarizes the results obtained (RFV signal), with the different combinations of antibodies used in capture or detection, on three dilutions of the purified native annexin A3 neutrophil (100, 25 and 3 ng / mL ). Table 1 Biotinylated revelation antibody Capture antibodies clone Dilution ANXA3 ng / ml TGC42 TGC43 TGC44 9C6B4 TGC42 0 - 22 11 17 3 - 44 20 78 25 - 238 89 500 100 - 859 297 1837 TGC43 0 106 - 39 63 3 111 - 36 82 25 144 - 56 257 100 232 - 86 914 TGC44 0 21 9 - 12 3 32 19 - 19 25 98 89 - 74 100 303 309 - 261 9C6B4 0 51 42 33 - 3 65 122 39 - 25 201 744 117 - 100 590 3525 357 - 5C5B10 0 20 12 93 9 3 5244 3669 5870 39 25 10480 9874 10429 249 100 11310 11095 11168 1139 13A12G4H2 0 26 21 18 19 3 2298 676 3733 23 25 9655 8468 10208 21 100 11380 10995 11332 21 6D9D10 0 12 23 16 9 3 11 23 14 9 25 12 23 18 19 100 18 41 43 27 1F10A6 0 45 9 14 14 3 159 207 194 17 25 4014 6728 5680 13 100 10489 10931 10688 15

Le tableau 2 ci-dessous résume les résultats obtenus décrits dans le tableau 1, mais avec une grille de lecture différente, pour faciliter l'analyse et l'interprétation :

  • Si RFV à 3 ng/mL < 1000 alors « - »
  • Si RFV à 3 ng/mL > 1000 alors « + »
  • Si RFV à 25 ng/mL > 3000 alors « ++ »
  • Si (RFV à 100 ng/mL > 9000) et (3000 < RFV à 25 ng/mL < 9000) alors « +++ »
  • Si RFV à 100 ng/mL et 25 ng/mL > 9000 alors « ++++ »
Tableau 2 *mAb de détection biotinylé *mAb de capture TGC42 TGC43 TGC44 9C6B4 5C5B10 13A12G4H2 1F10A6 6D9D10 TGC42 - - - - ++++ ++++ +++ - TGC43 - - - + ++++ +++ +++ - TGC44 - - - - ++++ ++++ +++ - 9C6B4 + - - - + - - - mAb = anticorps monoclonal Table 2 below summarizes the results obtained described in Table 1, but with a different reading grid, to facilitate analysis and interpretation:
  • If RFV at 3 ng / mL <1000 then "-"
  • If RFV at 3 ng / mL> 1000 then "+"
  • If RFV at 25 ng / mL> 3000 then "++"
  • If (RFV at 100 ng / mL> 9000) and (3000 <RFV at 25 ng / mL <9000) then "+++"
  • If RFV at 100 ng / mL and 25 ng / mL> 9000 then "++++"
Table 2 * biotinylated detection mAb * capture mAb TGC42 TGC43 TGC44 9C6B4 5C5B10 13A12G4H2 1F10A6 6D9D10 TGC42 - - - - ++++ ++++ +++ - TGC43 - - - + ++++ +++ +++ - TGC44 - - - - ++++ ++++ +++ - 9C6B4 + - - - + - - - mAb = monoclonal antibody

Comme cela ressort du tableau ci-dessus, il existe 9 combinaisons d'anticorps monoclonaux complémentaires qui permettent de réaliser un dosage ELISA de type sandwich de l'ANXA3, avec une dynamique de signal très satisfaisante (couples « +++ » et « ++++ »). D'autres combinaisons d'anticorps monoclonaux complémentaires sont possibles, à savoir 9C6B4 / TGC42, 9C6B4 / TGC43 et 9C6B4 / 5C5B10, mais ces solutions ne sont pas suffisamment robustes d'un point de vue analytique et n'ont pas la sensibilité analytique suffisante pour être utilisées dans l'urine.As can be seen from the table above, there are 9 combinations of complementary monoclonal antibodies that make it possible to perform a sandwich-type ELISA assay of ANXA3, with a very satisfactory signal dynamics ("+++" and "+" pairs). +++ "). Other combinations of complementary monoclonal antibodies are possible, namely 9C6B4 / TGC42, 9C6B4 / TGC43 and 9C6B4 / 5C5B10, but these solutions are not sufficiently robust from an analytical point of view and do not have sufficient analytical sensitivity to be used in the urine.

En capture, les anticorps monoclonaux TGC42, TGC43 et TGC44 ont des performances équivalentes vis-à-vis de l'Annexine 3.In capture, the monoclonal antibodies TGC42, TGC43 and TGC44 have equivalent performances with respect to Annexin 3.

En détection, les anticorps monoclonaux 5C5B10 et 13A12G4H2 ont aussi des performances équivalentes vis-à-vis de l'Annexine 3, alors l'anticorps monoclonal 1F10A6 donne des signaux satisfaisants mais plus faibles qu'avec les anticorps monoclonaux 5C5B10 et 13A12G4H2.In detection, the monoclonal antibodies 5C5B10 and 13A12G4H2 also have equivalent performances with respect to Annexin 3, whereas the monoclonal antibody 1F10A6 gives satisfactory but weaker signals than with the monoclonal antibodies 5C5B10 and 13A12G4H2.

Sélection des anticorps monoclonaux anti-ANXA3 selon leur capacité à détecter l'ANXA3 prostatiqueSelection of anti-ANXA3 monoclonal antibodies according to their ability to detect prostatic ANXA3

La complémentarité et la capacité des anticorps anti-ANXA3 à détecter l'ANXA3 prostatique a été analysée en utilisant la technique de l'ELISPOT. Il s'agit d'une variante de la technique ELISA qui détecte directement les sécrétions des cellules en culture. Les cellules humaines WPE1-NB26 (ATCC Cat No. CRL-2852), issues d'une lignée épithéliale prostatique les RWPE-1 (ATCC Cat No. CRL-11609) ont été choisies comme modèle d'expression de l'ANXA3 humaine produite par la prostate. Ces cellules ont été transformées en cellules cancéreuses par un traitement au MNU (N-methyl-N-nitrosourea) (10). Elles sont cultivées dans du milieu K-SFM (Gibco) supplémenté avec 5 ng/mL d'EGF (Epidermal Growth Factor) et 0,05 mg/mL de BPE (Bovin Pituitary Extract). Les lignées sont incubées à 37°C avec 5% de CO2.The complementarity and capacity of anti-ANXA3 antibodies to detect prostatic ANXA3 was analyzed using the ELISPOT technique. This is a variant of the ELISA technique that directly detects secretions from cells in culture. WPE1-NB26 human cells (ATCC Cat No. CRL-2852), derived from a prostatic epithelial line RWPE-1 (Cat ATCC No. CRL-11609) were chosen as the expression model of human ANXA3 produced. by the prostate. These cells were transformed into cancer cells by treatment with MNU (N-methyl-N-nitrosourea) (10). They are cultured in K-SFM medium (Gibco) supplemented with 5 ng / ml of EGF (Epidermal Growth Factor) and 0.05 mg / ml of BPE (Bovin Pituitary Extract). Lines are incubated at 37 ° C with 5% CO 2 .

Les anticorps monoclonaux de capture (TGC42, TGC43, TGC44, 5C5B10, 13A12G4H2, 9C6D9, 1F10A6) ont été adsorbés sur des plaques 96 puits MultiScreen™ HTS (Millipore, Cat no MSIP4510) à une concentration de 1 µg/puits dans du PBS stérile pendant une nuit à 4°C. Les plaques sont ensuite lavées au PBS et saturées avec du milieu de culture contenant 10% de sérum de veau foetal (SVF). Parallèlement, les cellules sont comptées, puis diluées et distribuées à raison 1000 cellules par puits. Les plaques sont incubées 20 h à 37°C et 5% de CO2, puis vidées. Les cellules restantes sont alors lysées par un traitement à l'eau glacée pendant 10 minutes. Les plaques sont ensuite lavées avec du PBS contenant 0,05% de Tween-20. Les anticorps de révélation biotinylés (TGC42, TGC43, TGC44, 5C5B10, 13A12G4H2, 9C6D9, 1F10A6) sont ajoutés à 0,1 µg/puits dilués en PBS-1% BSA-0,05% Tween-20 et incubés pendant 2h à température ambiante. Après plusieurs lavages, les spots sont révélés par l'ajout d'extravidine-phophatase alcaline (Sigma, Cat No. E2636) pendant une heure à température ambiante puis du substrat le 5 bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT, Biorad, Cat No. 170-6432).The monoclonal capture antibodies (TGC42, TGC43, TGC44, 5C5B10, 13A12G4H2, 9C6D9, 1F10A6) were adsorbed on 96-well MultiScreen ™ HTS plates (Millipore, Cat No. MSIP4510) at a concentration of 1 μg / well in sterile PBS. overnight at 4 ° C. The plates are then washed with PBS and saturated with culture medium containing 10% fetal calf serum (FCS). In parallel, the cells are counted, then diluted and distributed at 1000 cells per well. The plates are incubated for 20 hours at 37 ° C. and 5% CO 2 , and then emptied. The remaining cells are then lysed by treatment with ice water for 10 minutes. The plates are then washed with PBS containing 0.05% Tween-20. The biotinylated revelation antibodies (TGC42, TGC43, TGC44, 5C5B10, 13A12G4H2, 9C6D9, 1F10A6) are added at 0.1 μg / well diluted in PBS-1% BSA-0.05% Tween-20 and incubated for 2 hours at room temperature. room. After several washes, the spots are revealed by the addition of alkaline extravidine-phophatase (Sigma, Cat No. E2636) for one hour at room temperature. then bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium substrate (BCIP / NBT, Biorad, Cat No. 170-6432).

La sécrétion d'Annexine A3 par les cellules WPE1-NB26 est mesurée qualitativement par l'observateur. Le nombre de spots observés est reporté sur une échelle de - à ++++ (Tableau 3). La meilleure solution pour détecter l'Annexine A3 d'origine prostatique est d'apparier un anticorps parmi TGC42, TGC43 ou TGC44 avec un anticorps choisi parmi 13A12G4H2 et 1F10A6. Les anticorps choisis peuvent être utilisés indifféremment en capture ou en détection, la seule condition est que l'on associe un anticorps de chaque groupe.The secretion of Annexin A3 by WPE1-NB26 cells is qualitatively measured by the observer. The number of spots observed is plotted on a scale of - to ++++ (Table 3). The best solution for detecting prostatic Annexin A3 is to match one of TGC42, TGC43 or TGC44 with an antibody selected from 13A12G4H2 and 1F10A6. The selected antibodies can be used indifferently in capture or detection, the only condition is that one antibody is associated with each group.

Les résultats sont résumés dans le tableau 3 ci-dessous. Tableau 3 *mAb de détection biotinylé *mAab de capture TGC42 TGC43 TGC44 9C6B4 5C5B10 13A12G4H2 1F10A6 TGC42 - - - + ++ ++++ ++++ TGC43 - - - + ++ ++++ ++++ TGC44 - - - + ++ ++++ ++++ 9C6B4 - - - - - - - 5C5B10 - - - + - - - 13A12G4H2 ++++ ++++ ++++ + - - - 1F10A6 +++ ++++ +++ + - - - *mAab = anticorps monoclonal De façon très surprenante, l'annexine A3 sécrétée par une lignée prostatique cancéreuse est moins bien détectée lorsque l'on associe un anticorps choisis parmi TGC42, TGC43 et TGC44 en capture et l'anticorps 5C5B10 en détection. Or, avec l'immunodosage VIDAS® réalisé sur l'annexine A3 purifiée des neutrophiles une telle différence entre les anticorps 5C5B10 et 13A12G4H2 n'était pas observée, comme montré dans le tableau 2.The results are summarized in Table 3 below. Table 3 * biotinylated detection mAb * capture mAab TGC42 TGC43 TGC44 9C6B4 5C5B10 13A12G4H2 1F10A6 TGC42 - - - + ++ ++++ ++++ TGC43 - - - + ++ ++++ ++++ TGC44 - - - + ++ ++++ ++++ 9C6B4 - - - - - - - 5C5B10 - - - + - - - 13A12G4H2 ++++ ++++ ++++ + - - - 1F10A6 +++ ++++ +++ + - - - * mAab = monoclonal antibody Surprisingly, the annexin A3 secreted by a prostatic cancerous line is less well detected when an antibody selected from TGC42, TGC43 and TGC44 is combined in capture and the 5C5B10 antibody in detection. Now, with VIDAS ® immunoassay performed on annexin A3 purified neutrophils such a difference between 5C5B10 and 13A12G4H2 antibody was not observed, as shown in Table 2.

Détection de l'Annexine A3 dans les urines exprimées par les patients après un toucher rectalDetection of Annexin A3 in urine expressed by patients after digital rectal examination

Six échantillons d'urine exprimée après le toucher rectal ont été obtenus et traitées selon le procédé décrit dans la demande de brevet WO2007/141043 et par (11). La durée moyenne de la palpation de la prostate était de 10 secondes, au lieu des 20 secondes indiquées dans la demande WO2007/141043 .Six urine specimens expressed after the digital rectal examination were obtained and treated according to the method described in the patent WO2007 / 141043 and by (11). The average duration of the prostate palpation was 10 seconds instead of the 20 seconds indicated in the request WO2007 / 141043 .

L'ANXA3 contenue dans ces différents échantillons biologiques a été quantifiée avec chacun des neuf dosages ELISA sélectionnés. Les anticorps de détection biotinylés ont été dilués à 0,5 µg/mL pour les monoclonaux 5C5B10 et 13A12G4H2, à 1µg/mL pour le clone 1F10A6. Pour chaque dosage ELISA, une courbe de calibration a été établie en dosant une gamme de concentration de la protéine Annexine A3 native purifiée (Arodia). La courbe de calibration a été tracée en reportant en abscisse le logarithme en base dix de la concentration et en ordonnée le signal mesuré par le Vidas® en RFV. La concentration d'ANXA3 présent dans l'échantillon biologique a été calculée en interpolant la concentration correspondante au signal RFV lu par le Vidas®, à l'aide de modèles mathématiques de régression non linéaire comme un polynôme du troisième ordre ou un modèle 4-PL, bien connus de l'homme du métier. Les résultats sont présentas dans le tableau 4 ci dessous. Tableau 4 Doses d'annexine A3 en ng/mL selon le format du dosage ELISA (Anticorps de Capture - Anticorps de Détection) P* TGC42-5C5B10 TGC43-5C5B10 TGC44-5C5B10 TGC42-13A12G4H2 TGC43-13A12G4H 2 TGC44-13A12G4H 2 TGC42-1F10A6 TGC43-1F10A6 TGC44-1F10A6 A 90 99 123 0,1 0,1 0,1 0,1 0,1 0,1 B 5 2 5 9 11 13 13 14 14 C 2 1 2 6 8 8 9 9 10 D 11 3 13 78 80 81 77 86 97 E 22 7 28 110 144 154 120 136 149 F 1100 810 2100 0,0 0,0 0,0 0,0 0,0 0,0 P* = patient The ANXA3 contained in these different biological samples was quantified with each of the nine selected ELISA assays. The biotinylated detection antibodies were diluted to 0.5 μg / ml for the 5C5B10 and 13A12G4H2 monoclonals, to 1 μg / ml for the 1F10A6 clone. For each ELISA assay, a calibration curve was established by assaying a concentration range of the purified native Annexin A3 protein (Arodia). The calibration curve was plotted by plotting on the abscissa the logarithm in the base ten of the concentration and on the ordinate the signal measured by Vidas ® in RFV. The concentration of ANXA3 present in the biological sample was calculated by interpolating the concentration corresponding to the RFV signal read by the Vidas®, using mathematical nonlinear regression models such as a third-order polynomial or a 4- model. PL, well known to those skilled in the art. The results are shown in Table 4 below. Table 4 Annexin A3 doses in ng / mL according to ELISA assay format (Capture Antibody - Detection Antibody) P * TGC42-5C5B10 TGC43-5C5B10 TGC44-5C5B10 TGC42-13A12G4H2 TGC43-13A12G4H 2 TGC44-13A12G4H 2 TGC42-1F10A6 TGC43-1F10A6 TGC44-1F10A6 AT 90 99 123 0.1 0.1 0.1 0.1 0.1 0.1 B 5 2 5 9 11 13 13 14 14 VS 2 1 2 6 8 8 9 9 10 D 11 3 13 78 80 81 77 86 97 E 22 7 28 110 144 154 120 136 149 F 1100 810 2100 0.0 0.0 0.0 0.0 0.0 0.0 P * = patient

Les résultats des dosages ELISA Vidas® de l'ANXA3 urinaire chez les six patients sont récapitulés dans le tableau 4 et représentés dans la Figure 1. Ces résultats sont extrêmement surprenants et mettent en évidence une complexité de l'ANXA3 urinaire jusque-là insoupçonnée. En effet, pour un échantillon donné, les 9 formats de test ELISA ne rendent pas toujours la même dose. Cette différence est liée à l'anticorps de détection : pour un échantillon donné et un anticorps de détection donné, les doses rendues sont très similaires, voire identiques quelque soit l'anticorps de capture (TGC42, TGC43 ou TGC44). La corrélation entre les doses obtenues par les formats utilisant TGC42 et celles obtenues par les formats utilisant TGC43 est de 0,956 (r de Spearman, p-value<0,0001). La corrélation entre les doses mesurées par les formats utilisant TGC42 et celle obtenue par les formats utilisant TGC44 est de 0,994 (r de Spearman, p-value<0,0001). Les 3 anticorps de capture se comportent de manière très similaire. Ils ne sont pas la cause des différences observées.The results of the Vidas® ELISA assays for ANXA3 in all six patients are summarized in Table 4 and shown in the table below. Figure 1 . These results are extremely surprising and highlight a complexity of ANXA3 urinary hitherto unsuspected. Indeed, for a given sample, the 9 ELISA test formats do not always make the same dose. This difference is bound to the detection antibody: for a given sample and a given detection antibody, the rendered doses are very similar, or even identical regardless of the capture antibody (TGC42, TGC43 or TGC44). The correlation between the doses obtained by the formats using TGC42 and those obtained by the formats using TGC43 is 0.956 (Spearman r, p-value <0.0001). The correlation between the doses measured by the formats using TGC42 and that obtained by the formats using TGC44 is 0.994 (Spearman r, p-value <0.0001). The 3 capture antibodies behave very similarly. They are not the cause of the observed differences.

Les concentrations d'Annexine A3 calculées avec les ELISA utilisant en détection les anticorps 13A12G4H2 ou 1F10A6 sont également corrélées (r de Spearman r = 0.999, p-value<0,0001). La pente de la droite calculée en mettant en abscisse les doses obtenues par les ELISA utilisant 13A12G4H2 et en ordonnée celles obtenues par les ELISA utilisant 1F10A6 est de 1 : ces deux anticorps rendent des doses identiques. A l'inverse, il n'existe aucune corrélation entre les doses calculées avec l'anticorps 5C5B10 utilisé en détection et celles calculées avec le 13A12G4H2.Annexin A3 concentrations calculated with ELISA using 13A12G4H2 or 1F10A6 antibodies were also correlated (Spearman's r = 0.999, p-value <0.0001). The slope of the line calculated by placing on the abscissa the doses obtained by the ELISA using 13A12G4H2 and on the ordinate those obtained by the ELISA using 1F10A6 is 1: these two antibodies make identical doses. Conversely, there is no correlation between the doses calculated with the 5C5B10 antibody used in detection and those calculated with 13A12G4H2.

En fonction de cette analyse réalisée sur des urines exprimées, les 9 dosages ELISA peuvent être divisés en 2 groupes. Le groupe 1 correspond aux combinaisons des anticorps de capture TGC42, TGC43 ou TGC44 avec les clones 13A12G4H2 ou 1F10A6 utilisés en détection. Le groupe 2 correspond aux combinaisons des anticorps de capture TGC42, TGC43 ou TGC44 avec le monoclonal 5C5B10 utilisé en détection. Comme chacun de ces anticorps est bien spécifique de l'ANXA3, les deux groupes d'ELISA mesurent une information biologique différente. L'analyse en ELISPOT présentée ci dessus suggère que les ELISA du groupe 1 utilisant les clones 13A12G4H2 ou 1F10A6 sont plus adaptées à détecter l'ANXA3 d'origine prostatique.Based on this analysis performed on expressed urine, the 9 ELISA assays can be divided into 2 groups. Group 1 corresponds to the combinations of capture antibodies TGC42, TGC43 or TGC44 with clones 13A12G4H2 or 1F10A6 used in detection. Group 2 corresponds to the combinations of capture antibodies TGC42, TGC43 or TGC44 with the 5C5B10 monoclonal used in detection. Since each of these antibodies is specific for ANXA3, both ELISA groups measure different biological information. The ELISPOT assay presented above suggests that Group 1 ELISA using 13A12G4H2 or 1F10A6 clones are more suitable for detecting prostatic ANXA3.

Exemple 2 Caractérisation de l'annexine A3 urinaireExample 2 Characterization of Urinary Annexin A3

Huit échantillons d'urine exprimée post-toucher rectal ont été obtenus selon le procédé décrit dans l'exemple 1, transportés de l'hôpital au laboratoire et soumis aux analyses décrites dans les 4h qui suivent la collecte. Le dosage TGC44/13A12G4H2 (appelé uniquement 13A12G4H2 par la suite) a été choisi comme dosage prototype du groupe 1 défini dans l'exemple 1. De la même façon, le dosage TGC44/5C5B10 (appelé uniquement 5C5B10 par la suite) a été choisi comme dosage prototype représentant le groupe 2.Eight urine samples expressed after digital rectal examination were obtained according to the method described in Example 1, transported from the hospital to the laboratory and subjected to the analyzes described in the 4 hours following the collection. The TGC44 / 13A12G4H2 assay (called only 13A12G4H2 thereafter) was chosen as the assay Prototype Group 1 defined in Example 1. Similarly, TGC44 / 5C5B10 assay (called only 5C5B10 thereafter) was chosen as the prototype assay representing the group 2.

Analyse de l'urine exprimée par filtrations successivesAnalysis of urine expressed by successive filtrations

Les urines fraichement récoltées ont subit des filtrations successives :

  • Une première filtration sur une membrane avec des pores de 0,45 µm de diamètre (Millex), le filtrat est ensuite récupéré et un aliquot mis de côté pour dosages : FT 0,45 µm.
  • Le filtrat FT 0,45 µm est filtré sur une membrane de 0,22 µm (Millex) et un aliquot mis de côté pour dosages : FT 0,22 µm.
  • Le filtrat FT 0,22 µm est filtré de nouveau sur une membrane de 0,02 µm (Millipore) cette fois, dont la taille des pores retient les exosomes urinaires, c'est le FT 0,02 µm.
The freshly harvested urine has undergone successive filtrations:
  • A first filtration on a membrane with pores of 0.45 μm in diameter (Millex), the filtrate is then recovered and an aliquot set aside for assays: FT 0.45 microns.
  • The 0.45 μm FT filtrate is filtered on a membrane of 0.22 μm (Millex) and an aliquot set aside for assays: FT 0.22 μm.
  • The 0.22 μm FT filtrate is filtered again on a membrane of 0.02 μm (Millipore) this time, the pore size of which retains the urinary exosomes, the FT 0.02 μm.

Chacune de ces fractions est ensuite dosée avec les 2 formats de test ELISA prototype sur automate Vidas®. Les doses d'Annexine A3 mesurées dans chacune des fractions sont exprimées en pourcentage de la dose initiale d'Annexine A3 urinaire avant la première filtration (=100%). Les résultats sont représentés dans la Figure 2. La diminution de la dose entre deux filtrats successifs indique que des particules biologiques contenant de l'ANXA3 ont été retenues sur la membrane de filtration. Ainsi, selon des principes bien connus de l'homme du métier, une filtration sur 0,45 µm retient les cellules et les débris cellulaires (12, 13), une filtration sur 0,22 µm retient certains organelles et des fractions subcellulaires et une filtration sur 0,02 µm retient les exosomes (14-16). Cette diminution est bien observée pour tous les échantillons, avec les deux procédés de dosage utilisés (5C5B10 et 13A12G4H2). Dans la grande majorité des urines exprimées (6/8), environ 20 à 40% de l'ANXA3 se trouve associée à des débris cellulaires ou autres fractions subcellulaires. Les 60 à 80% restant sont éliminés par filtration sur 0,02 µm, et sont donc associés à des exosomes. Sur les 8 échantillons d'urine exprimée testés, de l'ANXA3 soluble (qui passe à travers un filtre de 0,02 µm) n'a été retrouvée que dans un unique échantillon avec le dosage 5C5B10 et dans deux échantillons avec le dosage 13A12G4H2.Each of these fractions is then assayed with the 2 prototype ELISA test formats on Vidas® PLC. The doses of Annexin A3 measured in each of the fractions are expressed as a percentage of the initial dose of urinary Annexin A3 before the first filtration (= 100%). The results are represented in the Figure 2 . The decrease in the dose between two successive filtrates indicates that biological particles containing ANXA3 were retained on the filtration membrane. Thus, according to principles well known to those skilled in the art, filtration on 0.45 microns retains cells and cellular debris (12, 13), a 0.22 micron filtration retains certain organelles and subcellular fractions and a filtration on 0.02 μm retains the exosomes (14-16). This decrease is well observed for all samples, with the two assay methods used (5C5B10 and 13A12G4H2). In the vast majority of urines expressed (6/8), approximately 20 to 40% of ANXA3 is associated with cellular debris or other subcellular fractions. The remaining 60 to 80% are removed by filtration over 0.02 μm, and are therefore associated with exosomes. Of the 8 tested urine specimens tested, soluble ANXA3 (which passes through a 0.02 μm filter) was found only in a single sample with the 5C5B10 assay and in two samples with the 13A12G4H2 assay. .

Analyse de l'urine exprimée par centrifugations successivesAnalysis of urine expressed by successive centrifugations

Les mêmes urines fraichement récoltées ont également été fractionnées par des centrifugations successives :

  • Les urines sont centrifugées une première fois à 800 g pendant 5 min, le surnageant est récupéré et un aliquot mis de côté pour dosages : SN 800.
  • Le surnageant urinaire issu de la centrifugation à 800 g est centrifugé à 12 000 g pendant 7 min, le surnageant est récupéré et un aliquot mis de côté pour dosages : SN 12 000.
  • Le surnageant urinaire obtenu après centrifugation à 12 000 g est ensuite ultracentrifugé à 150 000 g sur la nuit à 4°C afin de précipiter les exosomes urinaires dans le culot. Le surnageant récolté est le SN ultra.
The same freshly collected urine was also fractionated by successive centrifugations:
  • The urine is centrifuged a first time at 800 g for 5 min, the supernatant is recovered and an aliquot set aside for assays: SN 800.
  • The urine supernatant resulting from the centrifugation at 800 g is centrifuged at 12,000 g for 7 min, the supernatant is recovered and an aliquot set aside for assays: SN 12,000.
  • The urine supernatant obtained after centrifugation at 12,000 g is then ultracentrifuged at 150,000 g overnight at 4 ° C to precipitate urinary exosomes in the pellet. The harvested supernatant is the ultra SN.

Chacune de ces fractions est ensuite dosée avec les 2 formats de test ELISA prototype sur automate Vidas®. Les doses d'Annexine A3 mesurées dans chacune des fractions sont exprimées en pourcentage de la dose initiale d'Annexine A3 urinaire avant toute centrifugation (NC=100%). Les résultats sont représentés dans la Figure 3. La diminution de la dose entre deux surnageants successifs indique que des particules biologiques contenant de l'ANXA3 ont été séparées dans le culot lors de la centrifugation. Ainsi, selon des principes bien connus de l'homme du métier, une centrifugation à 800 g culote la plupart des cellules humaines, une centrifugation à 12 000 g culote les débris cellulaire résiduels et certains organelles et une ultracentrifugation sur la nuit culote toutes les particules subcellulaires, y compris les vésicules de type exosomes (17, 18). Toute protéine qui reste dans le surnageant après une ultracentrifugation est considérée comme étant soluble (19). Cette diminution est bien observée pour tous les échantillons, avec les deux procédés de dosage utilisés (5C5B10 et 13A12G4H2). Dans la grande majorité des urines exprimées (6/8), environ 20 à 25% de l'ANXA3 se trouve associée à des débris cellulaires ou autres fractions subcellulaires. Les 75 à 80% restant ne sont précipités que par ultracentrifugation sur la nuit, et sont donc associés à des particules de type exosomes. Sur les 8 échantillons d'urine exprimée testés, de l'ANXA3 soluble (qui reste dans le surnageant après une nuit d'ultracentrifugation) n'a été retrouvée que dans les deux échantillons déjà identifiés comme contenant de l'ANXA3 soluble avec l'analyse exposée au paragraphe 1, et ce avec les deux techniques de dosage 5C5B10 et 13A12G4H2.Each of these fractions is then assayed with the 2 prototype ELISA test formats on Vidas® PLC. The doses of Annexin A3 measured in each of the fractions are expressed as a percentage of the initial dose of urinary Annexin A3 prior to any centrifugation (NC = 100%). The results are represented in the Figure 3 . Decreasing the dose between two successive supernatants indicates that ANXA3-containing biological particles were separated in the pellet during centrifugation. Thus, according to principles well known to those skilled in the art, 800 g centrifugation culminates most human cells, a centrifugation at 12 000 g culminates residual cell debris and some organelles and ultracentrifugation on the night culminates all particles subcellular, including exosome-type vesicles (17, 18). Any protein that remains in the supernatant after ultracentrifugation is considered soluble (19). This decrease is well observed for all samples, with the two assay methods used (5C5B10 and 13A12G4H2). In the vast majority of urines expressed (6/8), approximately 20 to 25% of ANXA3 is associated with cellular debris or other subcellular fractions. The remaining 75 to 80% are precipitated only by ultracentrifugation at night, and are therefore associated with exosome-like particles. Of the 8 urine specimens tested, soluble ANXA3 (which remains in the supernatant after overnight ultracentrifugation) was found only in the two samples already identified as containing soluble ANXA3 with the analysis described in paragraph 1, with the two assay techniques 5C5B10 and 13A12G4H2.

Une nouvelle série de 10 urines fraîchement récoltées a été analysée en modifiant légèrement les conditions de centrifugation. La centrifugation à 800 g a été de 10 min. et celle à 12 000 g de 30 min. Les résultats de cette deuxième expérience de centrifugations successives sont présentées en Figure 4 et confirment les observations réalisées lors de la première expérience (Figure 3). De plus, ils montrent que le dosage 13A12G4H2 reconnaît plus fréquemment que le dosage 5C5B10 l'ANXA3 associé aux exosomes.A new series of 10 freshly harvested urine was analyzed by slightly modifying the centrifugation conditions. Centrifugation at 800 g was 10 min. and that at 12,000 g of 30 min. The results of this second experiment of successive centrifugations are presented in Figure 4 and confirm the observations made during the first experiment ( Figure 3 ). In addition, they show that the 13A12G4H2 assay recognizes more frequently than the 5C5B10 ANXA3 assay associated with exosomes.

Globalement les résultats des fractionnements par filtration et par centrifugation sont extrêmement concordants et indiquent que la majorité de l'ANXA3 à doser se trouve associée à des exosomes, mais pas uniquement. Nous avons montré que l'ANXA3 se trouve aussi associée à des particules de taille plus importante comme des débris cellulaires, et qu'elle pouvait également être sous forme soluble. La préparation d'échantillon pour une analyse en Western blot permet de solubiliser et de dénaturer les protéines et ainsi de diminuer cette complexité, ce qui peut en partie expliquer les difficultés rencontrées jusque-là pour trouver un procédé de dosage ELISA qui soit utilisable dans les fluides biologiques et en particulier l'urine. Bien entendu, le procédé de traitement des urines exprimées recueillies, les conditions de conservation (température, tampon) sont autant de facteurs qui peuvent faire varier la répartition de l'ANXA3 dans les différentes fractions urinaires où elle peut être présente.Overall, the filtration and centrifugation fractionation results are extremely consistent and indicate that the majority of the ANXA3 to be assayed is associated with exosomes, but not only. We have shown that ANXA3 is also associated with larger particles such as cell debris, and that it can also be in soluble form. The sample preparation for a Western blot analysis makes it possible to solubilize and denature the proteins and thus to reduce this complexity, which can partly explain the difficulties encountered so far in finding an ELISA assay method that can be used in the biological fluids and in particular urine. Of course, the method of treatment of the expressed urine collected, the storage conditions (temperature, buffer) are all factors that can vary the distribution of ANXA3 in the various urinary fractions where it may be present.

Exemple 3 : Détermination des épitopes reconnus par les anticorps monoclonaux anti-ANXA3Example 3 Determination of Epitopes Recognized by Anti-ANXA3 Monoclonal Antibodies Expression des 4 domaines « annexin repeat » de l'Annexine A3 sous forme recombinante et détermination des domaines répétés reconnus par les anticorps monoclonaux.Expression of the 4 annexin repeat domains of Annexin A3 in recombinant form and determination of the repeated domains recognized by the monoclonal antibodies.

Comme tous les membres de la famille des Annexines, l'Annexine A3 contient dans sa séquence protéique des domaines appelés « annexin repeat ». Ces domaines répétés sont au nombre de 4 et caractérisent la famille. Afin de déterminer le domaine répété reconnu par chacun des anticorps monoclonaux, ces domaines ont été exprimés sous une forme recombinante. Une séquence de 8 histidines a été rajoutée à la partie N-terminale de chaque domaine afin de permettre la purification par chromatographie d'affinité métal-chélate. Le tableau 5 récapitule les séquences protéiques des constructions de recombinants permettant d'exprimer chacun des domaines répétés de façon isolée. Tableau 5 Domaine Réel a Exprimé b Séquence protéique D1 27-87 19-89

Figure imgb0001
D2 99-159 92-160
Figure imgb0002
D3 183-243 171-245
Figure imgb0003
D4 258-318 252-323
Figure imgb0004
a Réel : Etendue du domaine répété, du premier au dernier acide aminé, selon la base de données UniProtKB (http://www.uniprot.org).
b Exprimé : Etendue de la construction contenant le domaine répété, numérotation des acides aminés selon UniProtKB. Les constructions contiennent quelques acides aminés supplémentaires aux extrémités N et C terminales du domaine, afin de ne pas interrompre les hélices alpha et leur permettre de se former.
Like all the members of the Annexin family, Annexin A3 contains in its protein sequence domains called annexin repeat. These repeated domains are four in number and characterize the family. To determine the repeated domain recognized by each of the monoclonal antibodies, these domains were expressed in a recombinant form. A sequence of 8 histidines was added to the N-terminal part of each domain to allow purification by metal-chelate affinity chromatography. Table 5 summarizes the protein sequences of the recombinant constructs making it possible to express each of the repeated domains in isolation. Table 5 Field Real a Expressed b Protein sequence D1 27-87 19-89
Figure imgb0001
D2 99-159 92-160
Figure imgb0002
D3 183-243 171-245
Figure imgb0003
D4 258-318 252-323
Figure imgb0004
a Real: Extent of repeated domain, from first to last amino acid, according to the UniProtKB database ( http://www.uniprot.org ).
b Expressed: Extent of the construction containing the repeated domain, numbering of amino acids according to UniProtKB. The constructs contain some extra amino acids at the N- and C-termini of the domain, so as not to interrupt the alpha helices and allow them to form.

Les séquences d'acides nucléiques correspondant aux séquences protéiques des domaines D1, D2, D3, D4 ont été obtenues par synthèse chimique par la société Geneart. Ces séquences nucléiques ont été optimisées pour favoriser l'expression des protéines en Escherichia coli. Les fragments d'ADN ont été clonés entre les sites Nco I et Xba I du vecteur d'expression procaryote pMRCH79 (dérivé du pMR78, bioMérieux). Les plasmides ainsi obtenus ont été transformés dans les bactéries BL21 (DE3) (Stratagene). Les cultures pour produire les différents domaines sont réalisées à 37°C sous agitation dans du milieu 2-YT (Invitrogen). L'induction est effectuée avec 0,5 mM d'IPTG (isopropyl beta-1-thiogalactosidase). Les culots bactériens sont directement repris dans le tampon d'échantillons des gels NuPAGE Novex (Invitrogen) en suivant le mode opératoire fourni avec les gels, en conditions réduites. La séparation des protéines est réalisée en gel NuPAGE Novex Bis-Tris 4-12%. L'analyse en Western blot de la réactivité des anticorps monoclonaux pour les différents domaines de l'annexine A3 est réalisée en utilisant un substrat chemiluminescent, selon le procédé décrit dans la demande de brevet WO 2009/019365 par exemple, bien connu de l'homme du métier. Les anticorps à tester ont été utilisés à une dilution de 10 µg/mL. Le temps d'exposition a été de 100 secondes, sauf indication contraire.The nucleic acid sequences corresponding to the protein sequences of the D1, D2, D3 and D4 domains were obtained by chemical synthesis by Geneart. These nucleic sequences have been optimized to promote the expression of proteins in Escherichia coli. The DNA fragments were cloned between the Nco I and Xba I sites of the prokaryotic expression vector pMRCH79 (derived from pMR78, bioMérieux). The plasmids thus obtained were transformed into BL21 (DE3) bacteria (Stratagene). The cultures to produce the different domains are carried out at 37 ° C. with stirring in 2-YT medium (Invitrogen). Induction is performed with 0.5 mM IPTG (isopropyl beta-1-thiogalactosidase). The bacterial pellets are directly taken up in the sample buffer of the NuPAGE Novex gels (Invitrogen) following the procedure provided with the gels under reduced conditions. Protein separation is performed in NuPAGE Novex Bis-Tris 4-12% Gel. Western blot analysis of the reactivity of the monoclonal antibodies for the different domains of annexin A3 is produced using a chemiluminescent substrate, according to the process described in the patent application WO 2009/019365 for example, well known to those skilled in the art. The antibodies to be tested were used at a dilution of 10 μg / ml. The exposure time was 100 seconds unless otherwise indicated.

Chaque anticorps anti-ANXA3 a été testé avec les recombinants exprimant les 4 domaines répétés de l'ANXA3 ; les résultats de cette analyse Western blot sont présentés dans la Figure 5. Les anticorps monoclonaux TGC42, TGC43, TGC44 et 5C5B10 sont spécifiques du domaine D1. Les anticorps monoclonaux 13A12G4H2 et 1F10A6 sont dirigés contre le domaine D4. Quant aux anticorps 9C6D4 et 6D9D10, ils ne reconnaissent aucun des 4 domaines répétés, ce qui indique très probablement que leurs épitopes sont situés en dehors des domaines répétés de l'ANXA3.Each anti-ANXA3 antibody was tested with recombinants expressing the 4 repeated ANXA3 domains; the results of this Western blot analysis are presented in the Figure 5 . The monoclonal antibodies TGC42, TGC43, TGC44 and 5C5B10 are specific for the D1 domain. Monoclonal antibodies 13A12G4H2 and 1F10A6 are directed against the D4 domain. As for the 9C6D4 and 6D9D10 antibodies, they do not recognize any of the 4 repeat domains, which most likely indicates that their epitopes are located outside the repeated domains of ANXA3.

Analyse fine des épitopes reconnus par les anticorps anti-ANXA3Fine analysis of epitopes recognized by anti-ANXA3 antibodies

La détermination des épitopes a été effectuée avec la technique du Spotscan d'après Frank et Döring (20), qui est décrite en détail dans la demande de brevet WO 2009/019365 . Dans ce but, la totalité de la séquence protéique de l'annexine A3 a été reproduite sur une membrane de nitrocellulose sous forme de peptides de 12 acides aminés chevauchants, décalés de 2 acides aminés. Puis dans une seconde synthèse, la séquence d'ANXA3 a été reproduite sous forme de peptides de 15 acides aminés chevauchants, décalés d'un acide aminé. L'immunoréactivité de ces membranes de peptides chevauchants a été testée avec les anticorps anti-ANXA3.The determination of epitopes has been made with the Spotscan technique according to Frank and Döring (20), which is described in detail in the patent application. WO 2009/019365 . For this purpose, the entire protein sequence of Annexin A3 was reproduced on a nitrocellulose membrane in the form of peptides of 12 overlapping amino acids, shifted by 2 amino acids. Then in a second synthesis, the ANXA3 sequence was reproduced as overlapping amino acid peptides, staggered with an amino acid. The immunoreactivity of these overlapping peptide membranes was tested with anti-ANXA3 antibodies.

Ainsi, il a été possible de délimiter de façon plus fine les épitopes de 5 anticorps monoclonaux anti-ANXA3 parmi les 8 étudiés. Les épitopes déduits de la comparaison des séquences des peptides chevauchants reconnus sont récapitulés dans le tableau 6. L'épitope minimal est la séquence minimale nécessaire pour avoir une reconnaissance de l'anticorps, avec un signal plus ou moins intense. L'épitope optimal est la séquence idéale permettant la meilleure reconnaissance possible de l'anticorps, incluant ou identique à l'épitope minimal. Nos résultats confirment que TGC42 et TGC43 sont bien dirigés contre un épitope unique, celui qui est décrit initialement dans la demande WO 2010/034825 . De façon surprenante, l'anticorps 5C5B10 définit un nouvel épitope qui n'était pas décrit dans l'art antérieur. Les anticorps 6D9D10 et 9C6D4 sont spécifiques de l'extrémité N-terminale de la protéine, comme le monoclonal TGC7 de la demande WO 2007/141043 . Les anticorps monoclonaux TGC44, 13A12G4H2 et 1F10A6 ne présentent aucune réactivité en Spotscan, même sur une membrane portant des peptides de 20 acides aminés de long. Ils possèdent probablement des épitopes conformationnels ou au moins semi-conformationnels dont les structures ne sont pas assez bien reproduites par des peptides de synthèse. Tableau 6 Anticorps Domaine Epitope optimal a Epitope minimal b TGC42 D1 SNAQRQLIVKEYQAAYG (49-65) (SEQ ID: 10) LIVKEYQAAYG (55-65) (SEQ ID: 11) TGC43 D1 IVKEYQAAYGKE (56-67) (SEQ ID: 12) KEYQAAYG (58-65) (SEQ ID: 13) 5C5B10 D1 DLSGHFEHL (75-83) (SEQ ID: 14) LSGHFEH (76-82) (SEQ ID: 15) 6D9D10 N-term ASIWVGHRGTVRDYPDFSPS (2-21) (SEQ ID: 16) SIWVGHRGTVRDYPDFSP (3-20) (SEQ ID: 17) 9C6D4 N-term YPDF (15-18) (SEQ ID: 18) YPDF (15-18) SEQ ID: 18) a Epitope optimal : Séquence idéale permettant la meilleure reconnaissance possible de l'anticorps (incluant ou identique à l'épitope minimal).
b Epitope minimal : Séquence minimale nécessaire pour avoir une reconnaissance de l'anticorps (signal plus ou moins intense).
Thus, it has been possible to more finely delineate anti-ANXA3 monoclonal antibody epitopes among the 8 studied. The epitopes deduced from the comparison of the sequences of the recognized overlapping peptides are summarized in Table 6. The minimal epitope is the minimum sequence necessary to have a recognition of the antibody, with a more or less intense signal. The optimal epitope is the ideal sequence for the best possible recognition of the antibody, including or identical to the minimal epitope. Our results confirm that TGC42 and TGC43 are well directed against a single epitope, the one that is originally described in the application WO 2010/034825 . Surprisingly, the 5C5B10 antibody defines a new epitope that was not described in the prior art. The antibodies 6D9D10 and 9C6D4 are specific for the N-terminus of the protein, as the monoclonal TGC7 of the application WO 2007/141043 . The monoclonal antibodies TGC44, 13A12G4H2 and 1F10A6 show no Spotscan reactivity, even on a membrane carrying peptides 20 amino acids long. They probably have conformational or at least semi-conformational epitopes whose structures are not sufficiently well reproduced by synthetic peptides. Table 6 Antibody Field Optimal epitope has Minimal epitope b TGC42 D1 SNAQRQLIVKEYQAAYG (49-65) (SEQ ID: 10) LIVKEYQAAYG (55-65) (SEQ ID: 11) TGC43 D1 IVKEYQAAYGKE (56-67) (SEQ ID: 12) KEYQAAYG (58-65) (SEQ ID: 13) 5C5B10 D1 DLSGHFEHL (75-83) (SEQ ID: 14) LSGHFEH (76-82) (SEQ ID: 15) 6D9D10 N-term ASIWVGHRGTVRDYPDFSPS (2-21) (SEQ ID: 16) SIWVGHRGTVRDYPDFSP (3-20) (SEQ ID: 17) 9C6D4 N-term YPDF (15-18) (SEQ ID: 18) YPDF (15-18) SEQ ID: 18) a Optimal epitope: Ideal sequence allowing the best possible recognition of the antibody (including or identical to the minimal epitope).
b Minimum epitope: Minimum sequence necessary to have recognition of the antibody (more or less intense signal).

Localisation précise de l'épitope de l'anticorps TGC44 par la technique NovatopeAccurate localization of the TGC44 antibody epitope by the Novatope technique

Le système Novatope (Merck, Cat No. 69279) est une technologie permettant l'analyse d'une protéine dans le but de sélectionner des domaines contenant des épitopes. La méthode est fondée sur la création d'une banque de clones bactériens, chacun exprimant une fragment de la protéine, coupée aléatoirement. Ces clones sont analysés par immunorévélation avec l'anticorps que l'on cherche à caractériser. Le séquençage de l'ADN des clones positifs permet de déduire la séquence protéique d'un fragment contenant l'épitope. La technique a été appliquée en suivant le mode opératoire fourni avec le kit.The Novatope system (Merck, Cat No. 69279) is a technology for the analysis of a protein for the purpose of selecting domains containing epitopes. The method is based on the creation of a library of bacterial clones, each expressing a fragment of the protein, cut randomly. These clones are analyzed by immunorevealing with the antibody that is to be characterized. DNA sequencing of the positive clones makes it possible to deduce the protein sequence from a fragment containing the epitope. The technique was applied following the procedure provided with the kit.

Ainsi, deux clones réagissant avec l'anticorps TGC44 ont pu être isolés et séquencés. Le clone 2J7 exprime la séquence KEYQAAYGKELKDDLKGDLSGHFEHLMVALVTPPAVFD (SEQ ID : 19) qui correspond aux résidus 58-95 de l'ANXA3. Le clone 2Z13 exprime la séquence QKAIRGIGTDEKMLISILTERSNAQRQLIVKEYQAAYGKELKDDLKGDLSGHFEHL (SEQ ID : 20) qui correspond aux résidus 28-83 de l'ANXA3. La partie commune entre les séquences de ces deux clones sont les résidus 58-83 de l'Annexine A3, soit la séquence KEYQAAYGKELKDDLKGDLSGHFEHL (SEQ ID : 21). De plus, les anticorps TGC44 et 5C5B10 étant complémentaires (voir exemple 1), leurs deux épitopes ne peuvent être chevauchants, on peut donc retirer les acides aminés correspondants à l'épitope de l'anticorps 5C5B10, soit DLSGHFEHL (75-83) (SEQ ID : 14). Donc l'épitope de l'anticorps TGC44 est compris dans la séquence KEYQAAYGKELKDDLKG (58-74) (SEQ ID : 22). Il s'agit de la même région que celle reconnue par TGC42 et TGC43. Par contre, le fait que l'anticorps TGC44 ne présente pas de réactivité en Spotscan indique une contrainte conformationnelle pour la reconnaissance. TGC44 est capable de se fixer sur son épitope uniquement lorsque la séquence KEYQAAYGKELKDDLKG (58-74) (SEQ ID : 22) est fusionnée du côté N-terminal à une séquence longue d'au moins 30 résidus. Ainsi, le domaine répété recombinant D1, dont la séquence est identifiée en SEQ ID NO: 6, les clones 2J7 et 2Z13 fusionnés à une protéine porteuse ou encore le fragment recombinant vANA-7 décrit demande WO 2010/034825 sont tous reconnus par le clone TGC44. A l'inverse, le fragment recombinant vANA-3, à qui il manque les 34 premiers acides aminés N-terminaux, n'est pas reconnu (Figure 6)Thus, two clones reacting with the TGC44 antibody could be isolated and sequenced. Clone 2J7 expresses the sequence KEYQAAYGKELKDDLKGDLSGHFEHLMVALVTPPAVFD (SEQ ID: 19) which corresponds to residues 58-95 of ANXA3. Clone 2Z13 expresses the sequence QKAIRGIGTDEKMLISILTERSNAQRQLIVKEYQAAYGKELKDDLKGDLSGHFEHL (SEQ ID: 20) which corresponds to residues 28-83 of ANXA3. The common part between the sequences of these two clones are the residues 58-83 of Annexin A3, that is the sequence KEYQAAYGKELKDDLKGDLSGHFEHL (SEQ ID: 21). In addition, the TGC44 and 5C5B10 antibodies being complementary (see Example 1), their two epitopes can not be overlapping, one can thus remove the amino acids corresponding to the epitope of the antibody 5C5B10, or DLSGHFEHL (75-83) ( SEQ ID: 14). Thus, the epitope of the TGC44 antibody is included in the sequence KEYQAAYGKELKDDLKG (58-74) (SEQ ID: 22). This is the same region as that recognized by TGC42 and TGC43. On the other hand, the fact that the TGC44 antibody does not exhibit Spotscan reactivity indicates a conformational constraint for the recognition. TGC44 is able to bind to its epitope only when the sequence KEYQAAYGKELKDDLKG (58-74) (SEQ ID: 22) is fused on the N-terminal side to a long sequence of at least 30 residues. Thus, the recombinant repetitive domain D1, whose sequence is identified in SEQ ID NO: 6, clones 2J7 and 2Z13 fused to a carrier protein or the recombinant fragment vANA-7 described requires WO 2010/034825 are all recognized by clone TGC44. In contrast, the recombinant fragment vANA-3, which lacks the first 34 N-terminal amino acids, is not recognized ( Figure 6 )

Localisation précise de l'épitope des anticorps 13A12G4H2 et 1F10A6Accurate localization of the 13A12G4H2 and 1F10A6 antibody epitope

L'expérience présentée en Figure 5 montre que les épitopes des anticorps 13A12G4H2 et 1F10A6 sont contenus dans le domaine D4 de l'annexine A3. Douze protéines recombinantes ont été construites par mutagénèse dirigée afin d'améliorer le « mapping » des anticorps dirigés contre le domaine D4. Il s'agit de la séquence complète de l'ANXA3 native mature (aa 2-323), fusionnée du côté N-terminal à un tag histidine (séquence non mutée). La mutagenèse par PCR a été utilisée afin d'introduire des mutations Alanine en positions 253, 257, 260, 263, 265, 268, 270, 274, 306, 311 et 317 de la séquence protéique (GeneArt Mutagenesis Service, Invitrogen). Cette technique connue sous le nom d'Alanine scanning permet d'évaluer un par un l'importance de la contribution de chaque résidu acide aminé muté de l'Annexine A3 à la fixation des anticorps 13A12G4H2 et 1F10A6. Tous ces fragments d'ADN ont été clonés dans le vecteur pMRCH79 dérive du vecteur pMR78, puis transformés dans les bactéries BL21 (DE3). La production et la purification des protéines ont été réalisées selon les techniques bien connues de l'homme du métier et qui ont été citées au début de l'exemple 3.The experience presented in Figure 5 shows that the epitopes of the 13A12G4H2 and 1F10A6 antibodies are contained in the D4 domain of annexin A3. Twelve recombinant proteins were constructed by site-directed mutagenesis to enhance the "mapping" of antibodies against the D4 domain. This is the complete sequence of mature native ANXA3 (aa 2-323), fused on the N-terminal side to a histidine tag (unmutated sequence). PCR mutagenesis was used to introduce Alanine mutations at positions 253, 257, 260, 263, 265, 268, 270, 274, 306, 311 and 317 of the protein sequence (GeneArt Mutagenesis Service, Invitrogen). This technique known as Alanine scanning allows to evaluate one by one the importance of the contribution of each mutated amino acid residue of Annexin A3 to the binding of antibodies 13A12G4H2 and 1F10A6. All these DNA fragments were cloned into the vector pMRCH79 derived from the vector pMR78, then transformed into BL21 bacteria (DE3). The production and purification of the proteins were carried out according to the techniques well known to those skilled in the art and which were mentioned at the beginning of Example 3.

Ces 12 protéines (11 mutants et 1 témoin non mutée) ont ensuite été utilisées pour évaluer la capacité de fixation des anticorps 13A12G4H2 et 1F10A6 dans un ELISA au format sandwich, utilisant l'anticorps TGC44 en capture. L'anticorps de détection 5C5B10 dont l'épitope est dans le domaine D1 et de ce fait, ne devrait pas être affecté par les mutations du domaine D4 est utilisé comme témoin. Les résultats sont présentés dans la Figure 7. Le graphe représente le « signal fold change » (en ordonnée) de chaque mutation (en abscisse). Le « signal fold change » correspond au log2 (signal protéine mutée / signal protéine non mutée). Plus la valeur absolue du signal fold change est élevée, plus les mutations pour lesquelles cette variation est observée affectent la fixation de l'anticorps. Ainsi, pour 5C5B10, le signal fold change est toujours autour de 0, indiquant qu'aucune des mutations testées ne perturbe la fixation de ce monoclonal. Par contre, pour 13A12G4H2 et 1F10A6, il a été possible d'identifier des acides aminés dont la mutation empêche ou perturbe de façon très significative la fixation. Il s'agit des positions 260, 263, 265 et 306. La mutation de la position 270 a un impact mesurable mais moins important que pour les positions précédentes. Ceci montre que les acides aminés 260, 263, 265 et 306 d'ANXA3 appartiennent à l'épitope reconnu par les anticorps monoclonaux 13A12G4H2 et 1F10A6, les deux résidus les plus importants dans l'interaction antigène-anticorps étant la Lysine (K) en position 263 et l'Acide Aspartique (D) en position 306.These 12 proteins (11 mutants and 1 non-mutated control) were then used to evaluate the binding capacity of the 13A12G4H2 and 1F10A6 antibodies in a sandwich ELISA, using the TGC44 antibody in capture. The 5C5B10 detection antibody whose epitope is in the D1 domain and therefore should not be affected by the D4 domain mutations is used as a control. The results are presented in the Figure 7 . The graph represents the "fold change signal" (on the ordinate) of each mutation (on the abscissa). The "fold change signal" corresponds to log 2 (mutated protein signal / non-mutated protein signal). The higher the absolute value of the fold change signal, the more the mutations for which this variation is observed affect the binding of the antibody. Thus, for 5C5B10, the fold change signal is always around 0, indicating that none of the mutations tested disturb the binding of this monoclonal. On the other hand, for 13A12G4H2 and 1F10A6, it was possible to identify amino acids whose mutation prevents or disrupts the fixation very significantly. These are positions 260, 263, 265 and 306. The mutation of position 270 has a measurable but less significant impact than for the previous positions. This shows that the amino acids 260, 263, 265 and 306 of ANXA3 belong to the epitope recognized by the monoclonal antibodies 13A12G4H2 and 1F10A6, the two most important residues in the antigen-antibody interaction being Lysine (K). position 263 and Aspartic Acid (D) at position 306.

Exemple 4 : Spécificité analytique des formats de dosage ELISA anti-ANXA3Example 4 Analytical Specificity of Anti-ANXA3 ELISA Assay Formats

Les Annexines sont une famille de protéines qui partagent des homologies de fonction et de séquence. Une interrogation BLAST effectuée sur la base de donnée UniProtKB, réduite aux séquences d'origine humaine, a permis d'identifier les protéines ayant la plus grande homologie de séquence avec l'Annexine A3. Il s'agit, dans l'ordre décroissant d'homologie, de l'annexine A4, l'annexine A11, l'annexine A6 et l'annexine A5.Annexins are a family of proteins that share function and sequence homologies. A BLAST query performed on the UniProtKB database, reduced to sequences of human origin, has made it possible to identify the proteins having the greatest sequence homology with Annexin A3. These are, in descending order of homology, annexin A4, annexin A11, annexin A6 and annexin A5.

Par conséquent, il était important de démontrer la spécificité des dosages ELISA vis-à-vis de l'Annexine A3 et l'absence de réaction croisée avec les autres membres de la famille. Comme les 3 anticorps TGC42, TGC43 et TGC44 sont dirigés contre le même épitope, le dosage TGC44/13A12G4H2 (appelé uniquement 13A12G4H2 par la suite) a été choisi comme dosage prototype du groupe 1 défini dans l'exemple 1. De la même façon, le dosage TGC44/5C5B10 (appelé uniquement 5C5B10 par la suite) a été choisi comme dosage prototype représentant le groupe 2. L'absence de réactivité croisée a été testée en utilisant des antigènes disponibles commercialement obtenus chez Abnova : annexine A1 (Cat. No. H00000301-P01), annexine A2 (Cat. No. H00000302-P01), annexine A4 (Cat. No. H00000307-P01), annexine A5 (Cat. No. H00000308-P01), annexine A6 (Cat. No. H00000309-P01) et annexine A11 (Cat. No. H00000311-P01). L'annexine A13 a été exprimée sous forme recombinante au laboratoire par clonage dans le vecteur d'expression pMRCH79 ; fusionnée à un tag histidine, puis purifiée par chromatographie d'affinité métal-chélate. Les protéines ont été diluées dans le tampon du puits X1 du VIDAS à une concentration de 12,5 µg/mL pour l'ELISA TGC44/5C5B10 et à une concentration de 16 µg/mL pour l'ELISA TGC44/13A12G4H2. Les résultats sont présentés dans la Figure 8. Les deux formats d'ELISA TGC44/13A12G4H2 et TGC44/5C5B10 sont tous les deux très spécifiques de l'Annexine A3 et ne présentent pas de réactivité croisée avec les autres annexines testées.Therefore, it was important to demonstrate the specificity of ELISA assays for Annexin A3 and the absence of cross-reactivity with other family members. Since the 3 antibodies TGC42, TGC43 and TGC44 are directed against the same epitope, the TGC44 / 13A12G4H2 assay (hereinafter called only 13A12G4H2) was chosen as the prototype group 1 assay defined in Example 1. In the same way, the TGC44 / 5C5B10 assay (hereinafter only referred to as 5C5B10) was chosen as the prototype assay representing group 2. The lack of cross-reactivity was tested using commercially available antigens obtained from Abnova: annexin A1 (Cat No. H00000301-P01), Annexin A2 (Cat No. H00000302-P01), Annexin A4 (Cat No. H00000307-P01), Annexin A5 (Cat No. H00000308-P01), Annexin A6 (Cat No. H00000309- P01) and annexin A11 (Cat No. H00000311-P01). Annexin A13 was expressed in recombinant form in the laboratory by cloning into the pMRCH79 expression vector; fused to a histidine tag, and then purified by metal-chelate affinity chromatography. The proteins were diluted in the VIDAS well X1 buffer at a concentration of 12.5 μg / mL for TGC44 / 5C5B10 ELISA and at a concentration of 16 μg / mL for TGC44 / 13A12G4H2 ELISA. The results are presented in the Figure 8 . Both TGC44 / 13A12G4H2 and TGC44 / 5C5B10 ELISA formats are both highly specific for Annexin A3 and show no cross-reactivity with the other annexins tested.

Exemple 5 : Détermination de l'affinité des anticorps anti-Annexine A3Example 5 Determination of the Affinity of Anti-Annexin A3 Antibodies

La technologie de résonance plasmonique de surface permet de visualiser en temps réel les interactions entre diverses biomolécules non marquées. Un des réactifs est fixé de manière spécifique sur un biocapteur (sensor chip) tandis que l'autre espèce impliquée dans l'interaction est dans un flux continu de tampon. Les mesures de résonance plasmonique de surface ont été effectuées en utilisant un Biacore T100. Les réactifs incluant le sensor chip CM5, l'anti-IgG de souris, spécifique du fragment Fc, obtenu chez le lapin (RAM Fc), et le kit de couplage via les amines pour l'immobilisation des anticorps ont tous été obtenus chez GE-Healthcare Bioscience AB.Surface plasmon resonance technology allows real-time visualization of interactions between various unlabeled biomolecules. One of the reagents is specifically attached to a sensor chip while the other species involved in the interaction is in a continuous stream of buffer. Surface plasmon resonance measurements have been performed using a Biacore T100. The reagents including the CM5 sensor chip, anti-mouse IgG, specific for the Fc fragment, obtained in the rabbit (RAM Fc), and the coupling kit via the amines for the immobilization of the antibodies were all obtained from GE -Healthcare Bioscience AB.

Afin d'étudier les caractéristiques cinétiques de fixation des anticorps anti-Annexine A3, ces derniers ont été immobilisés par capture sur le sensor chip, sur lequel, l'anticorps RAM Fc avait été couplé de façon covalente, au préalable. Les expériences de fixation ont été réalisées en tampon, HEPES, à 25°C, avec un débit de 30 µL/min. La modification du signal de résonnance en RU (Resonance Unit) permet de suivre en temps réel la fixation puis la dissociation des biomolécules à la surface du biocapteur. Dans un premier temps, l'anticorps monoclonal à étudier a été injecté dans le canal 2 pour obtenir un signal d'environ 250 RU. Ensuite, l'Annexine A3 (Arodia) a été injecté dans les canaux 1 et 2. Les durées d'association et de dissociation étaient respectivement de 5 et 15 minutes. Après mesure des réponses de résonance, la surface du biocapteur a été régénérée par un lavage avec du HCl 50 mM, à 10 µL /min pendant 120 secondes. Le même procédé de mesure a été répété pour chaque dilution de la protéine Annexine A3 ; au total 9 dilutions différentes de la protéine comprises entre 0 et 64 nM ont été analysées. Les sensorgrammes obtenus ont été tracés et analysés avec le logiciel dédié du Biacore™ T100, selon le modèle d'interaction 1:1. Les constantes cinétiques d'association (Kon) et de dissociation (Koff) ont été mesurées en utilisant les anticorps à une concentration de 3 µg/mL, sauf pour 13A12G4H2 et 1F10A6 qui ont été utilisés à 0,75 µg/mL afin de limiter l'impact du bruit de fond. L'affinité, représentée par la constante de dissociation (Kd), a été calculée (Kd=Koff/Kon).In order to study the kinetic binding characteristics of the anti-Annexin A3 antibodies, the latter were immobilized by capture on the sensor chip, on which the RAM Fc antibody had been previously covalently coupled. The binding experiments were performed in buffer, HEPES, at 25 ° C, with a flow rate of 30 μl / min. The modification of the resonance signal in RU (Resonance Unit) makes it possible to follow in real time the fixation and the dissociation of the biomolecules on the surface of the biosensor. Initially, the monoclonal antibody to be studied was injected into channel 2 to obtain a signal of approximately 250 RU. Subsequently, Annexin A3 (Arodia) was injected into channels 1 and 2. The association and dissociation times were respectively 5 and 15 minutes. After measuring the resonance responses, the surface of the biosensor was regenerated by washing with 50 mM HCl at 10 μL / min for 120 seconds. The same measurement method was repeated for each dilution of the Annexin A3 protein; in total 9 different protein dilutions between 0 and 64 nM were analyzed. The resulting sensorgrams were plotted and analyzed with the dedicated Biacore ™ T100 software, according to the 1: 1 interaction model. The kinetic constants of association (Kon) and dissociation (Koff) were measured using the antibodies at a concentration of 3 μg / mL, except for 13A12G4H2 and 1F10A6 which were used at 0.75 μg / mL in order to limit the impact of background noise. The affinity, represented by the dissociation constant (Kd), was calculated (Kd = Koff / Kon).

Pour chaque anticorps anti-Annexine A3, les graphiques représentant le signal de résonance en fonction du temps sont présentés dans la Figure 9. Les valeurs mesurées des constantes d'association et de dissociation, ainsi que la valeur calculée des constantes d'affinité sont indiquées dans le tableau 7.For each anti-Annexin A3 antibody, the graphs representing the resonance signal as a function of time are presented in FIG. Figure 9 . The measured values of the association and dissociation constants, as well as the calculated value of the affinity constants are shown in Table 7.

L'ensemble des anticorps anti-ANXA3 étudiés présente des affinités élevées, avec des Kd allant de 10-9 à 5 x 10-10 M. Il est possible de classer les anticorps en deux groupes distincts en fonction de leur Kd. Le premier groupe correspond aux anticorps 13A12G4H2, TGC42 et TGC44 dont la Kd est supérieure à 10-10 M, c'est le groupe des anticorps de très haute affinité. Le second groupe correspond aux anticorps 5C5B10, 1F10A6 et TGC43 dont la Kd est comprise entre 10-9 et 3,5 x 10-9 M, c'est le groupe des anticorps de haute affinité.The set of anti-ANXA3 antibodies studied has high affinities, with Kd values ranging from 10 -9 to 5 x 10 -10 M. The antibodies can be classified into two distinct groups. function of their Kd. The first group corresponds to antibodies 13A12G4H2, TGC42 and TGC44 whose Kd is greater than 10 -10 M, it is the group of very high affinity antibodies. The second group corresponds to the antibodies 5C5B10, 1F10A6 and TGC43 whose Kd is between 10 -9 and 3.5 x 10 -9 M, it is the group of high affinity antibodies.

Les trois anticorps utilisés en capture, TGC42, TGC43 et TGC44, ont des constantes cinétiques d'association équivalentes, donc des capacités de capture équivalentes. L'anticorps 13A12G4H2 a également une constante cinétique d'association comparable, confirmant ainsi sa capacité de capture montrée dans l'exemple 1. La constante cinétique d'association de l'anticorps 5C5B10 est d'environ 1 log plus bas que celle de TGC44, ce qui illustre sa moins bonne capacité de capture.The three antibodies used in capture, TGC42, TGC43 and TGC44, have equivalent kinetic association constants, and therefore equivalent capture capacities. The 13A12G4H2 antibody also has a comparable association kinetic constant, thus confirming its capture capacity shown in Example 1. The kinetic constant of the 5C5B10 antibody is approximately 1 log lower than that of TGC44. , which illustrates its lower catch capacity.

En ce qui concerne les constantes cinétiques de dissociation, il est aussi possible de diviser les anticorps anti-ANXA3 en 2 groupes. Le premier groupe contient TGC44, TGC42, 13A12G4H2 et 5C5B10 ; il est caractérisé par une constante cinétique de dissociation très faible, comprise entre 9 x 10-5 et 3.5 x 10-4 s-1. Une fois que la fixation antigène-anticorps a eu lieu, l'Annexine A3 est retenue par les anticorps de ce groupe et ne se dissocie pas. Le second groupe contient TGC43 et 1F10A6 ; il est caractérisé par une constante cinétique de dissociation plus élevée, de l'ordre du 10-3 s-1. Les anticorps de ce groupe, même s'ils arrivent à fixer l'Annexine A3, s'en dissocient bien plus rapidement. Ainsi, bien qu'ayant des constantes cinétiques d'association comparables, TGC42 et TGC44 sont de meilleurs anticorps de capture à utiliser pour la mise au point d'un dosage ELISA que TGC43. Tableau 7 Anticorps fixé sur le biocapteur Kon (M-1.s-1) Koff (s-1) Kd (M) 13A12G4H2 5,3E+05 2,7E-04 5,0E-10 TGC42 7,3E+05 1,0E-04 1,8E-10 TGC44 9,5E+05 9,7E-05 1,0E-10 5C5B10 9,8E+04 3,4E-04 3,4E-09 1F10A6 3,8E+05 1,1E-03 2,9E-09 TGC43 8,4E+05 1,1E-03 1,3E-09 With regard to the kinetic dissociation constants, it is also possible to divide the anti-ANXA3 antibodies into 2 groups. The first group contains TGC44, TGC42, 13A12G4H2 and 5C5B10; it is characterized by a very low dissociation kinetic constant, between 9 x 10 -5 and 3.5 x 10 -4 s -1 . Once antigen-antibody binding has occurred, Annexin A3 is retained by the antibodies in this group and does not dissociate. The second group contains TGC43 and 1F10A6; it is characterized by a higher dissociation kinetic constant, of the order of 10 -3 s -1 . Antibodies in this group, even if they succeed in attaching Annexin A3, dissociate much more rapidly. Thus, although having comparable kinetic association constants, TGC42 and TGC44 are better capture antibodies to be used for the development of an ELISA assay than TGC43. Table 7 Antibody fixed on the biosensor Kon (M -1 .s -1 ) Koff (s -1 ) Kd (M) 13A12G4H2 5,3E + 05 2.7E-04 5,0E-10 TGC42 7,3E + 05 1.0E-04 1,8E-10 TGC44 9,5E + 05 9,7E-05 1.0E-10 5C5B10 9,8E + 04 3.4E-04 3.4E-09 1F10A6 3,8E + 05 1,1E-03 2,9E-09 TGC43 8,4E + 05 1,1E-03 1.3E-09

Exemple 6 : Utilisation des dosages ELISA prototypes pour distinguer cancer et pas cancer dans des cohortes de patientsExample 6 Use of Prototype ELISA Assays to Differentiate Cancer and Not Cancer in Patient Cohorts

Afin d'analyser la capacité de l'Annexine A3 à distinguer les patients ayant un cancer de la prostate de ceux qui n'en ont pas, les dosages ELISA décrits dans l'exemple 1 ont été utilisés pour doser la quantité d'Annexine A3 présente dans les urines post-toucher rectal de ces patients.In order to analyze the ability of Annexin A3 to distinguish patients with prostate cancer from those who do not, the ELISA assays described in Example 1 were used to assay the amount of Annexin A3. present in the urine post-rectal examination of these patients.

Les patients inclus dans le groupe « Cancer » ont tous un cancer de la prostate avéré, dont le diagnostic a été confirmé par analyse histologique sur biopsie. Pour les patients inclus dans le groupe « Non cancer » ou contrôle, le cancer de la prostate a été exclus par biopsie également ; il s'agit dans la très grande majorité de patients ayant une hyperplasie bénigne de la prostate. La collecte et le traitement des échantillons d'urine post-toucher rectal ont été réalisés selon le procédé décrit dans l'exemple 1. Deux cohortes de patients ont été analysées : La cohorte #1 comprend 127 patients, 72 dans le groupe « Cancer » et 55 dans le groupe « Non cancer ». La cohorte #2 comprend 94 patients, 43 dans le groupe « Cancer » et 42 dans le groupe « Non cancer ». Pour les deux cohortes, il s'agit de patients ayant un taux de PSA sérique compris entre 2,5 et 10 ng/mL. C'est la zone grise du PSA, dans laquelle les performances cliniques du marqueur sont les moins bonnes.The patients included in the "Cancer" group all have proven prostate cancer, whose diagnosis was confirmed by histological analysis on a biopsy. For patients included in the "No cancer" or control group, prostate cancer was excluded by biopsy as well; the vast majority are patients with benign prostatic hyperplasia. The collection and treatment of post-digital rectal examination urine samples were carried out according to the method described in Example 1. Two cohorts of patients were analyzed: Cohort # 1 comprises 127 patients, 72 in the "Cancer" group and 55 in the "Non cancer" group. Cohort # 2 includes 94 patients, 43 in the "Cancer" group and 42 in the "Non cancer" group. For both cohorts, these are patients with serum PSA between 2.5 and 10 ng / mL. This is the gray area of PSA, in which the clinical performance of the marker is the worst.

Les premiers dosages ELISA réalisés sur les urines exprimées après le toucher rectal, décrits dans l'exemple 1 ont permis de mettre en évidence une complexité biologique insoupçonnée jusque là. Nous avons réussi à définir deux groupes de dosages ELISA qui mesurent une information biologique différente (partiellement redondant ou pas). Il est donc essentiel de déterminer quel groupe de dosages ELISA permet d'accéder à l'information biologique la plus pertinente, i.e. permet de discriminer au mieux entre les « Cancers » et les « Non cancers » dans un échantillonnage donné. Le dosage TGC44/13A12G4H2 (appelé uniquement 13A12G4H2 par la suite) a été choisi comme dosage prototype du groupe 1 défini dans l'exemple 1. De la même façon, le dosage TGC44/5C5B10 (appelé uniquement 5C5B10 par la suite) a été choisi comme dosage prototype représentant le groupe 2. La Figure 10 représente les valeurs obtenues pour chacune des cohortes #1 et #2 avec les deux formats de dosage ELISA Annexine A3 utilisés. La dose d'Annexine A3 a été normalisée par rapport à la valeur de la densité urinaire mesurée par la bandelette Combur 10 (Roche Cat. No. 04510062171), selon la formule dose normalisée=dose VIDAS/(densité urinaire-1) (21).The first ELISA assays carried out on the urine expressed after the digital rectal examination, described in Example 1, made it possible to highlight a biological complexity that was previously unsuspected. We have succeeded in defining two groups of ELISA assays that measure different biological information (partially redundant or not). It is therefore essential to determine which group of ELISA assays gives access to the most relevant biological information, ie it makes it possible to better discriminate between "Cancers" and "No cancers" in a given sample. The TGC44 / 13A12G4H2 assay (hereinafter called 13A12G4H2 only) was chosen as the prototype group 1 assay defined in Example 1. Similarly, the TGC44 / 5C5B10 assay (referred to only as 5C5B10 thereafter) was chosen as a prototype assay representing group 2. The Figure 10 represents the values obtained for each cohort # 1 and # 2 with both formats Annexin A3 ELISA assay used. The dose of Annexin A3 was normalized to the urinary density value measured by the Combur 10 strip (Roche Cat No. 04510062171), according to the standardized dose formula = VIDAS / dose (urinary density-1) (21). ).

Dans la première cohorte de patients (cohorte #1) les deux formats de dosages permettent de discriminer les patients ayant un cancer des contrôles. En effet, les doses d'Annexine A3 normalisées sont significativement plus basses dans le groupe « Cancer » que dans le groupe « Non cancer », quel que soit le format de dosage utilisé (p-value Mann-Whitney unilatéral est de 0,003 pour le dosage TGC44/5C5B10 et de 0,02 pour le dosage TGC44/13A12G4H2). Par contre dans la seconde cohorte (cohorte #2), c'est uniquement le format de dosage TGC44/13A12G4H2 qui permet de discriminer les patients ayant un cancer des contrôles. Comme pour la cohorte #1 et en accord avec l'étude réalisée par la technique de Western blot par Schostack et coll. (21), les doses d'Annexine A3 normalisées sont significativement plus basses dans le groupe « Cancer » que dans le groupe « Non cancer » en utilisant l'ELISA prototype TGC44/13A12G4H2 (p-value Mann-Whitney unilatéral est de 0.01). Par contre le prototype TGC44/5C5B10 est pris à dépourvu sur cette seconde cohorte et ne permet pas de discriminer les patients ayant un cancer. Le prototype TGC44/13A12G4H2 et de façon plus générale les formats ELISA du groupe 1 semblent donc être supérieurs pour discriminer les cancers de la prostate. Cette conclusion est concordante avec l'analyse ELISPOT décrite dans l'exemple 1 qui suggère que les ELISA du groupe 1 sont plus adaptées à détecter l'ANXA3 d'origine prostatique.In the first cohort of patients (cohort # 1) the two assay formats make it possible to discriminate cancer patients from controls. In fact, the standardized doses of Annexin A3 are significantly lower in the "Cancer" group than in the "Non cancer" group, regardless of the dosage format used (unilateral Mann-Whitney p-value is 0.003 for the TGC44 / 5C5B10 assay and 0.02 for the TGC44 / 13A12G4H2 assay). On the other hand, in the second cohort (cohort # 2), it is only the TGC44 / 13A12G4H2 assay format that makes it possible to discriminate patients with cancer from controls. As for cohort # 1 and in agreement with the Western blot study by Schostack et al. (21), standardized Annexin A3 doses were significantly lower in the "Cancer" group than in the "Non cancer" group using the prototype TGC44 / 13A12G4H2 ELISA (one-sided Mann-Whitney p-value was 0.01) . On the other hand, the prototype TGC44 / 5C5B10 is taken by surprise on this second cohort and does not make it possible to discriminate the patients having a cancer. The prototype TGC44 / 13A12G4H2 and, more generally, the Group 1 ELISA formats thus seem to be superior in discriminating prostate cancers. This conclusion is consistent with the ELISPOT assay described in Example 1 which suggests that Group 1 ELISA is more suitable for detecting prostatic ANXA3.

Exemple 7 : Effet de l'ion calcium et de l'EDTA sur les dosages 5C5B10 et 13A12G4H2Example 7 Effect of Calcium Ion and EDTA on Assays 5C5B10 and 13A12G4H2

Onze urines recueillies après le toucher rectal ont été dosées directement (sans traitement) ou après addition de 5 ou 25 mM de CaCl2 ou après addition de 5 ou 25 mM d'EDTA. Les résultats sont présentés en Figure 11. L'axe des ordonnées représente le ratio des doses avec traitement (Ca2+ ou EDTA) / dose sans traitement, et ce pour le dosage 5C5B10 et le dosage 13A12G4H2.L'addition de l'ion calcium dans les urines fait baisser les doses mesurées par le dosage 13A12G4H2 et ceci de façon progressive, plus la concentration de calcium ajoutée est augmentée, plus la dose mesurée est basse. Le dosage 5C5B10 est beaucoup moins affecté par la présence de calcium dans les urines, même si à haute concentration de calcium un léger effet est observé. Quant au traitement EDTA des urines, il n'a pas d'effet sur le dosage 13A12G4H2 mais pour le dosage 5C5B10, il provoque une très légère augmentation des doses mesurées. L'introduction d'EDTA ou d'un agent chélatant similaire dans les urines avant dosage, via les tampons ou sous forme solide, formulée ou non, permet donc d'améliorer les dosages, en particulier le dosage 13A12G4H2 en permettant de réduire l'effet de l'ion calcium sur les doses d'ANXA3.Eleven urine collected after the digital rectal examination were dosed directly (without treatment) or after addition of 5 or 25 mM CaCl 2 or after addition of 5 or 25 mM EDTA. The results are presented in Figure 11 . The y-axis represents the ratio of the doses with treatment (Ca 2+ or EDTA) / dose without treatment, for the 5C5B10 assay and the 13A12G4H2 assay. The addition of the calcium ion in the urine reduces the doses measured by the 13A12G4H2 assay and this in a progressive manner, plus the added calcium concentration is increased, the lower the measured dose. The 5C5B10 assay is much less affected by the presence of calcium in the urine, although at high calcium concentration a slight effect is observed. As for the EDTA treatment of the urine, it has no effect on the 13A12G4H2 assay but for the 5C5B10 assay, it causes a very slight increase in the measured doses. The introduction of EDTA or a similar chelating agent into the urine before dosing, via the buffers or in solid form, formulated or not, thus makes it possible to improve the assays, in particular the 13A12G4H2 assay by making it possible to reduce the effect of calcium ion on doses of ANXA3.

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    Figure imgb0030

Claims (26)

  1. A process for in vitro diagnosis of a prostate cancer, according to which a urine sample to be analysed is contacted with two antibodies, a capture antibody and a detection antibody, one of the two antibodies is directed against the first repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 1, and the other of the two antibodies is directed against the fourth repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 2.
  2. The process according to claim 1, in which the antibody directed against the first repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 17 consecutive amino acids of SEQ ID NO: 1.
  3. The process according to claim 1, in which the antibody directed against the first repeat domain of native human Annexin A3 is chosen from the antibodies directed against a polypeptide included in SEQ ID NO: 1, the amino acid sequence of which is selected from the following sequences:
    SNAQRQLIVKEYQAAYG (SEQ ID NO: 10),
    LIVKEYQAAYG (SEQ ID NO: 11)
    IVKEYQAAYGKE (SEQ ID NO: 12),
    KEYQAAYG (SEQ ID NO: 13),
    DLSGHFEHL (SEQ ID NO: 14),
    LSGHFEH (SEQ ID NO: 15),
    and
    KEYQAAYGKELKDDLKG (SEQ ID NO: 22), provided that the amino acid sequence SEQ ID NO: 22 is fused on the N-terminus side to a sequence of at least 30 amino acids.
  4. The process according to claim 1, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 50 consecutive amino acids of SEQ ID NO: 2.
  5. The process according to claim 4, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 45 consecutive amino acids of SEQ ID NO: 2.
  6. The process according to claim 4, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope which is included in an amino acid sequence corresponding to the amino acid sequence starting at residue 3 and ending at residue 49 of SEQ ID NO: 2.
  7. The process according to claim 4, 5 or 6, in which the epitope comprises in position 6 of SEQ ID NO: 2 a Lys residue.
  8. The process according to claim 4, 5 or 6, in which the epitope comprises in position 6 of SEQ ID NO: 2 a Lys residue and in position 49 of SEQ ID NO: 2 an Asp residue.
  9. The process according to claim 8, in which the epitope comprises in position 7 of SEQ ID NO: 2 a Gly residue, in position 8 of SEQ ID NO: 2 an Ile residue, and in position 9 of SEQ ID NO: 2 a Gly residue.
  10. The process according to claim 4, in which the epitope comprises in position 3 of SEQ ID NO: 2 an Arg residue, in position 6 of SEQ ID NO: 2 a Lys residue, in position 7 of SEQ ID NO: 2 a Gly residue, in position 8 of SEQ ID NO: 2 an Ile residue, in position 9 of SEQ ID NO: 2 a Gly residue, and in position 49 of SEQ ID NO: 2 an Asp residue.
  11. The process according to any one of the preceding claims, in which the antibody directed against the first repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 1, is the capture antibody and the antibody directed against the fourth repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 2, is the detection antibody.
  12. The process according to claim 1, in which the capture antibody and detection antibody are antibodies which exhibit a high affinity, with an affinity constant of at least 10-9.
  13. The process according to claim 1, in which the capture antibody and detection antibody are antibodies which exhibit a low dissociation constant of less than 2 x 10-3 s-1.
  14. An immunoassay kit for in vitro diagnosis of a prostate cancer in a urine sample to be analysed, comprising two antibodies, a capture antibody and a detection antibody, one of the two antibodies being directed against the first repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 1, and the other of the two antibodies being directed against the fourth repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 2.
  15. A kit according to claim 14, in which the antibody directed against the first repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 17 consecutive amino acids of SEQ ID NO: 1.
  16. A kit according to claim 14, in which the antibody directed against the first repeat domain of native human Annexin A3 is chosen from the antibodies directed against a polypeptide included in SEQ ID NO: 1, the amino acid sequence of which is selected from the following sequences:
    SNAQRQLIVKEYQAAYG (SEQ ID NO: 10),
    LIVKEYQAAYG (SEQ ID NO: 11)
    IVKEYQAAYGKE (SEQ ID NO: 12),
    KEYQAAYG (SEQ ID NO: 13),
    DLSGHFEHL (SEQ ID NO: 14),
    LSGHFEH (SEQ ID NO: 15),
    and
    KEYQAAYGKELKDDLKG (SEQ ID NO: 22), provided that the amino acid sequence SEQ ID NO: 22 is fused on the N-terminus side to a sequence of at least 30 amino acids.
  17. A kit according to claim 14, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 50 consecutive amino acids of SEQ ID NO: 2.
  18. A kit according to claim 17, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 45 consecutive amino acids of SEQ ID NO: 2.
  19. A kit according to claim 17, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope which is included in an amino acid sequence corresponding to the amino acid sequence starting at residue 3 and ending at residue 49 of SEQ ID NO: 2.
  20. A kit according to claim 17, 18 or 19, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, said epitope comprising in position 6 of SEQ ID NO: 2 a Lys residue.
  21. A kit according to claim 17, 18 or 19, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, said epitope comprising in position 6 of SEQ ID NO: 2 a Lys residue and in position 49 of SEQ ID NO: 2 an Asp residue.
  22. A kit according to claim 21, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, said epitope comprising in position 7 of SEQ ID NO: 2 a Gly residue, in position 8 of SEQ ID NO: 2 an Ile residue and in position 9 of SEQ ID NO: 2 a Gly residue.
  23. A kit according to claim 17, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, said epitope comprising in position 3 of SEQ ID NO: 2 an Arg residue, in position 6 of SEQ ID NO: 2 a Lys residue, in position 7 of SEQ ID NO: 2 a Gly residue, in position 8 of SEQ ID NO: 2 an Ile residue, in position 9 of SEQ ID NO: 2 a Gly residue, and in position 49 of SEQ ID NO: 2 an Asp residue.
  24. A kit according to any one of claims 14 to 23, comprising the capture antibody, which is directed against the first repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 1, and the detection antibody, which is directed against the fourth repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 2.
  25. A kit according to claim 14, in which the capture antibody and the detection antibody are antibodies which exhibit a high affinity, with an affinity constant of at least 10-9.
  26. A kit according to claim 14, in which the capture antibody and detection antibody are antibodies which exhibit a low dissociation constant of less than 2 x 10-3 s-1.
EP11811042.8A 2010-12-08 2011-12-07 Method and kit for the in vitro diagnosis of prostate cancer Not-in-force EP2649452B1 (en)

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FR1060221A FR2968767B1 (en) 2010-12-08 2010-12-08 METHOD AND KIT FOR IN VITRO DIAGNOSIS OF PROSTATE CANCER
PCT/FR2011/052893 WO2012076812A1 (en) 2010-12-08 2011-12-07 Method and case for the in vitro diagnosis of prostate cancer

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WO2013076222A1 (en) * 2011-11-23 2013-05-30 Proteosys Ag Differential annexin a3 measurements of serum and blood derivatives or fractions thereof for the diagnosis of prostate cancer
FR2991777B1 (en) * 2012-06-07 2015-08-21 Biomerieux Sa METHOD FOR DETECTING AND / OR ASSAYING APPENDIX A3 OF A MAMMAL IN THE BLOOD OR AT LEAST ONE OF ITS DERIVATIVES
FR3014198B1 (en) 2013-12-03 2017-03-03 Biomerieux Sa METHOD FOR ISOLATING EXOSOMES
AU2017324983A1 (en) * 2016-09-07 2019-04-18 Saksin Lifesciences Pvt Ltd Synthetic antibodies against VEGF and their uses

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US7608413B1 (en) * 2005-03-25 2009-10-27 Celera Corporation Kidney disease targets and uses thereof
EP1724586A3 (en) 2005-05-21 2007-07-04 ProteoSys AG Annexin for cancer risk assessment
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FR2919060B1 (en) 2007-07-19 2012-11-30 Biomerieux Sa METHOD OF DETERMINING EZRINE FOR IN VITRO DIAGNOSIS OF COLORECTAL CANCER
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